Breast cancer therapy based on hormone receptor status with nanoparticles comprising taxane

ABSTRACT

The present invention relates to methods and kits for the treatment of breast cancer based on hormone receptor status of progesterone receptor and estrogen receptor comprising the administration of a taxane alone, in combination with at least one other and other therapeutic agents, as well as other treatment modalities useful in the treatment of breast cancer. In particular, the invention relates to the use of nanoparticles comprising paclitaxel and albumin (such as Abraxane®) either alone or in combination with other chemotherapeutic agents or radiation, which may be used for the treatment of breast cancer which does not express estrogen receptor and/or progesterone receptor.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/519,126, filed Oct. 28, 2009, which is a National Phase filing under35 U.S.C. § 371 of International Application No. PCT/US2007/025645having an international filing date of Dec. 14, 2007, which claimspriority benefit to the providional application 60/875,004, filed Dec.14, 2006, the entire content of each of which is incorporated herein byreference.

TECHNICAL FIELD

The present invention relates to methods and kits for the treatment ofbreast cancer based on hormone receptor status of the progesteronereceptor and the estrogen receptor comprising the administration of ataxaric alone, in combination with at least one other and othertherapeutic agents, as well as other treatment modalities useful in thetreatment of breast cancer. In particular, the invention relates to theuse of nanoparticles comprising paclitaxel and albumin (such asAbraxane®) either alone or in combination with other chemotherapeuticagents or radiation, which may be used for the treatment of breastcancer which does not express the estrogen receptor and/or progesteronereceptor.

BACKGROUND

The failure of a significant number of tumors to respond to drug and/orradiation therapy is a serious problem in the treatment of cancer. Infact, this is one of the main reasons why many of the most prevalentforms of human cancer still resist effective chemotherapeuticintervention, despite certain advances in the field of chemotherapy.

Cancer is now primarily treated with one or a combination of three typesof therapies: surgery, radiation, and chemotherapy. Surgery is atraditional approach in which all or part of a tumor is removed from thebody. Surgery generally is only effective for treating the earlierstages of cancer. While surgery is sometimes effective in removingtumors located at certain sites, for example, in the breast, colon, andskin, it cannot be used in the treatment of tumors located in otherareas, inaccessible to surgeons, nor in the treatment of disseminatedneoplastic conditions such as leukemia. For more than 50% of cancerindividuals, by the time they are diagnosed they are no longercandidates for effective surgical treatment. Surgical procedures mayincrease tumor metastases through blood circulation during surgery. Mostof cancer individuals do not die from the cancer at the time ofdiagnosis or surgery, but rather die from the metastasis and therecurrence of the cancer.

Other therapies are also often ineffective. Radiation therapy is onlyeffective for individuals who present with clinically localized diseaseat early and middle stages of cancer, and is not effective for the latestages of cancer with metastasis. Radiation is generally applied to adefined area of the subject's body which contains abnormal proliferativetissue, in order to maximize the dose absorbed by the abnormal tissueand minimize the dose absorbed by the nearby normal tissue. However, itis difficult (if not impossible) to selectively administer therapeuticradiation to the abnormal tissue. Thus, normal tissue proximate to theabnormal tissue is also exposed to potentially damaging doses ofradiation throughout the course of treatment. There are also sometreatments that require exposure of the subject's entire body to theradiation, in a procedure called “total body irradiation”, or “TBI.” Theefficacy of radiotherapeutic techniques in destroying abnormalproliferative cells is therefore balanced by associated cytotoxiceffects on nearby normal cells. Because of this, radiotherapy techniqueshave an inherently narrow therapeutic index which results in theinadequate treatment of most tumors. Even the best radiotherapeutictechniques may result in incomplete tumor reduction, tumor recurrence,increasing tumor burden, and induction of radiation resistant tumors.

Chemotherapy involves the disruption of cell replication or cellmetabolism. Chemotherapy can be effective, but there are severe sideeffects, e.g., vomiting, low white blood cells (WBC), loss of hair, lossof weight and other toxic effects. Because of the extremely toxic sideeffects, many cancer individuals cannot successfully finish a completechemotherapy regime. Chemotherapy-induced side effects significantlyimpact the quality of life of the individual and may dramaticallyinfluence individual compliance with treatment. Additionally, adverseside effects associated with chemotherapeutic agents are generally themajor dose-limiting toxicity (DLT) in the administration of these drugs.For example, mucositis is one of the major dose limiting toxicity forseveral anticancer agents, including the antimetabolite cytotoxic agents5-FU, methotrexate, and antitumor antibiotics, such as doxorubicin. Manyof these chemotherapy-induced side effects if severe may lead tohospitalization, or require treatment with analgesics for the treatmentof pain. Some cancer individuals die from the chemotherapy due to poortolerance to the chemotherapy. The extreme side effects of anticancerdrugs are caused by the poor target specificity of such drugs. The drugscirculate through most normal organs of individuals as well as intendedtarget tumors. The poor target specificity that causes side effects alsodecreases the efficacy of chemotherapy because only a fraction of thedrugs is correctly targeted. The efficacy of chemotherapy is furtherdecreased by poor retention of the anti-cancer drugs within the targettumors.

Due to the severity and breadth of neoplasm, tumor and cancer, there isa great need for effective treatments of such diseases or disorders thatovercome the shortcomings of surgery, chemotherapy, and radiationtreatment.

Problems of Chemotherapeutic Agents

The drug resistance problem is a reason for the added importance ofcombination chemotherapy, as the therapy both has to avoid the emergenceof resistant cells and to kill pre-existing cells which are already drugresistant.

Drug resistance is the name given to the circumstance when a diseasedoes not respond to a treatment drug or drugs. Drug resistance can beeither intrinsic, which means the disease has never been responsive tothe drug or drugs, or it can be acquired, which means the disease ceasesresponding to a drug or drugs that the disease had previously beenresponsive to. Multidrug resistance (MDR) is a specific type of drugresistance that is characterized by cross-resistance of a disease tomore than one functionally and/or structurally unrelated drugs.Multidrug resistance in the field of cancer is discussed in greaterdetail in “Detoxification Mechanisms and Tumor Cell Resistance toAnticancer Drugs,” by Kuzmich and Tew, particularly section VII “TheMultidrug-Resistant Phenotype (MDR),”Med. Research Rev. 11(2): 185-217,(Section VII is at pp. 208-213) (1991); and in “Multidrug Resistance andChemosensitization: Therapeutic Implications for Cancer Chemotherapy,”by Georges, Sharom and Ling, Adv. in Pharmacology 21: 185-220 (1990).

One form of multi-drug resistance (MDR) is mediated by a membrane bound170-180 kD energy-dependent efflux pump designated as P-glycoprotein(P-gp). P-glycoprotein has been shown to play a major role in theintrinsic and acquired resistance of a number of human tumors againsthydrophobic, natural product drugs. Drugs that act as substrates for andare consequently detoxified by P-gp include the vinca alkaloids(vincristine and vinblastine), anthracyclines (Adriamycin), andepipodophyllotoxins (etoposide). While P-gp associated MDR is a majordeterminant in tumor cell resistance to chemotherapeutic agents, it isclear that the phenomenon of MDR is multifactorial and involves a numberof different mechanisms.

A major complication of cancer chemotherapy and of antiviralchemotherapy is damage to bone marrow cells or suppression of theirfunction. Specifically, chemotherapy damages or destroys hematopoieticprecursor cells, primarily found in the bone marrow and spleen,impairing the production of new blood cells (granulocytes, lymphocytes,erythrocytes, monocytes, platelets, etc.). Treatment of cancerindividuals with 5-fluorouracil, for example, reduces the number ofleukocytes (lymphocytes and/or granulocytes), and can result in enhancedsusceptibility of the individuals to infection. Many cancer individualsdie of infection or other consequences of hematopoietic failuresubsequent to chemotherapy. Chemotherapeutic agents can also result insubnormal formation of platelets which produces a propensity towardhemorrhage. Inhibition of erythrocyte production can result in anemia.For some cancer individuals, the risk of damage to the hematopoieticsystem or other important tissues frequently limits the opportunity forchemotherapy dose escalation of chemotherapy agents high enough toprovide good antitumor or antiviral efficacy. Repeated or high dosecycles of chemotherapy may be responsible for severe stem cell depletionleading to serious long-term hematopoietic sequelea and marrowexhaustion.

Prevention of, or protection from, the side effects of chemotherapywould be a great benefit to cancer individuals. For life-threateningside effects, efforts have concentrated on altering the dose andschedules of the chemotherapeutic agent to reduce the side effects.Other options are becoming available, such as the use of granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF),epidermal growth factor (EGF), interleukin 11, erythropoietin,thrombopoietin, megakaryocyte development and growth factor, pixykines,stem cell factor, FLT-ligand, as well as interleukins 1, 3, 6, and 7, toincrease the number of normal cells in various tissues before the startof chemotherapy (See Jimenez and Yunis, Cancer Research 52: 413-415(1992)). The mechanisms of protection by these factors, while not fullyunderstood, are most likely associated with an increase in the number ofnormal critical target cells before treatment with cytotoxic agents, andnot with increased survival of cells following chemotherapy.

Chemotherapeutic Targeting For Tumor Treatment

Both the growth and metastasis of solid tumors areangiogenesis-dependent (Folkman, J. Cancer Res. 46: 467-73 (1986);Folkman, J Nat. Cancer Inst. 82: 4-6 (1989); Folkman et al., “TumorAngiogenesis,” Chapter 10, pp. 206-32, in The Molecular Basis of Cancer,Mendelsohn et al., eds. (W. B. Saunders, 1995)). It has been shown, forexample, that tumors which enlarge to greater than 2 mm in diameter mustobtain their own blood supply and do so by inducing the growth of newcapillary blood vessels. After these new blood vessels become embeddedin the tumor, they provide nutrients and growth factors essential fortumor growth as well as a means for tumor cells to enter the circulationand metastasize to distant sites, such as liver, lung or bone (Weidner,New Eng. J. Med. 324(1): 1-8 (1991)). When used as drugs intumor-bearing animals, natural inhibitors of angiogenesis can preventthe growth of small tumors (O'Reilly et al., Cell 79: 315-28 (1994)).Indeed, in some protocols, the application of such inhibitors leads totumor regression and dormancy even after cessation of treatment(O'Reilly et al., Cell 88: 277-85 (1997)). Moreover, supplyinginhibitors of angiogenesis to certain tumors can potentiate theirresponse to other therapeutic regimes (e.g., chemotherapy) (see, e.g.,Teischer et al., Int J. Cancer 57: 920-25 (1994)).

Protein tyrosine kinases catalyze the phosphorylation of specifictyrosyl residues in various proteins involved in the regulation of cellgrowth and differentiation (A. F. Wilks, Progress in Growth FactorResearch (1990) 2: 97-111; S. A. Courtneidge, Dev. Suppl. (1993) 57-64;J. A. Cooper, Semin. Cell Biol. (1994) 5(6): 377-387; R. F. Paulson,Semin. Immunol. (1995) 7(4): 267-277; A. C. Chan, Curr. Opin. Immunol.(1996) 8(3): 394-401). Protein tyrosine kinases can be broadlyclassified as receptor (e.g., EGFr, c-erbB-2, c-met, tie-2, PDGFr, FGFr)or non-receptor (e.g., c-src, Ick, Zap70) kinases. Inappropriate oruncontrolled activation of many of these kinases, i.e., aberrant proteintyrosine kinase activity, for example by over-expression or mutation,has been shown to result in uncontrolled cell growth. For example,elevated epidermal growth factor receptor (EGFR) activity has beenimplicated in non-small cell lung, bladder and head and neck cancers,and increased c-erbB-2 activity in breast, ovarian, gastric andpancreatic cancers. Thus, inhibition of protein tyrosine kinases shouldbe useful as a treatment for tumors such as those outlined above.

Growth factors are substances that induce cell proliferation, typicallyby binding to specific receptors on cell surfaces. Epidermal growthfactor (EGF) induces proliferation of a variety of cells in vivo, and isrequired for the growth of most cultured cells. The EGF receptor is a170-180 kD membrane-spanning glycoprotein, which is detectable on a widevariety of cell types. The extracellular N-terminal domain of thereceptor is highly glycosylated and binds EGF antibodies thatselectively bind to EGFR. Agents that competitively bind to EGFR havebeen used to treat certain types of cancer, since many tumors ofmesodermal and ectodermal origin overexpress the EGF receptor. Forexample, the EGF receptor has been shown to be overexpressed in manygliomas, squamous cell carcinomas, breast carcinomas, melanomas,invasive bladder carcinomas and esophageal cancers. Attempts to exploitthe EGFR system for anti-tumor therapy have generally involved the useof monoclonal antibodies against the EGFR. In addition, studies withprimary human mammary tumors have shown a correlation between high EGFRexpression and the presence of metastases, higher rates ofproliferation, and shorter individual survival.

Herlyn et al., in U.S. Pat. No. 5,470,571, disclose the use ofradiolabeled Mab 425 for treating gliomas that express EGFR. Herlyn etal. report that anti-EGFR antibodies may either stimulate or inhibitcancer cell growth and proliferation. Other monoclonal antibodies havingspecificity for EGFR, either alone or conjugated to a cytotoxiccompound, have been reported as being effective for treating certaintypes of cancer. Bendig et al., in U.S. Pat. No. 5,558,864, disclosetherapeutic anti-EGFR Mab's for competitively binding to EGFR. Heimbrooket al., in U.S. Pat. No. 5,690,928, disclose the use of EGF fused to aPseudomonas species-derived endotoxin for the treatment of bladdercancer. Brown et al., in U.S. Pat. No. 5,859,018, disclose a method fortreating diseases characterized by cellular hyperproliforation mediatedby, inter alia, EGF.

Chemotherapeutic Modes of Administration

People diagnosed as having cancer are frequently treated with single ormultiple chemotherapeutic agents to kill cancer cells at the primarytumor site or at distant sites to where cancer has metastasized.Chemotherapy treatment is typically given either in a single or inseveral large doses or over variable times of weeks to months. However,repeated or high dose cycles of chemotherapy may be responsible forincreased toxicities and severe side effects.

New studies suggest that metronomic chemotherapy, the low-dose andfrequent administration of cytotoxic agents without prolonged drug-freebreaks, targets activated endothelial cells in the tumor vasculature. Anumber of preclinical studies have demonstrated superior anti-tumorefficacy, potent antiangiogenic effects, and reduced toxicity and sideeffects (e.g., myelosuppression) of metronomic regimes compared tomaximum tolerated dose (MID) counterparts (Bocci et al., Cancer Res 62:6938-6943 (2002); Bocci et al., PNAS 100(22): 12917-12922 (2003); andBertolini et al., Cancer Res 63(15): 4342-4346 (2003)). It remainsunclear whether all chemotherapeutic drugs exert similar effects orwhether some are better suited for such regimes than others.Nevertheless, metronomic chemotherapy appears to be effective inovercoming some of the major shortcomings associated with chemotherapy.

Chemotherapeutic Agents

Paclitaxel has been shown to have significant antineoplastic andanticancer effects in drug-refractory ovarian cancer and has shownexcellent antitumor activity in a wide variety of tumor models, and alsoinhibits angiogenesis when used at very low doses (Grant et al., Int. J.Cancer, 2003). The poor aqueous solubility of paclitaxel, however,presents a problem for human administration. Indeed, the delivery ofdrugs that are inherently insoluble or poorly soluble in an aqueousmedium can be seriously impaired if oral delivery is not effective.Accordingly, currently used paclitaxel formulations (e.g., Taxol®)require a Cremophor® to solubilize the drug. The presence of Cremophor®in this formulation has been linked to severe hypersensitivity reactionsin animals (Lorenz et al., Agents Actions 7: 63-67 (1987)) and humans(Weiss et al., J. Clin. Oncol. 8: 1263-68 (1990)) and consequentlyrequires premedication of individuals with corticosteroids(dexamethasone) and antihistamines. It was also reported that clinicallyrelevant concentrations of the formulation vehicle Cremophor® EL inTaxol® nullify the antiangiogenic activity of paclitaxel, suggestingthat this agent or other anticancer drugs formulated in Cremophor® ELmay need to be used at much higher doses than anticipated to achieveeffective metronomic chemotherapy (Ng et al., Cancer Res. 64: 821-824(2004)). As such, the advantage of the lack of undesirable side effectsassociated with low-dose paclitaxel regimes vs. conventional MTDchemotherapy may be compromised. See also U.S. Patent Pub. No.2004/0143004; WO00/64437.

Abraxane® is a Cremophor® EL-Free Nanoparticle Albumin-Bound Paclitaxel

Preclinical models have shown significant improvement in the safety andefficacy of Abraxane® compared with Taxol® (Desai et al.,EORTC-NCI-AACR, 2004) and in individuals with metastatic breast cancer(O'Shaughnessy et al., San Antonio Breast Cancer Symposium, Abstract#1122, December 2003). This is possibly due to the absence ofsurfactants (e.g., Cremophor® or Tween® 80, used in Taxol® andTaxotere®, respectively) in Abraxane®, and/or preferential utilizationof an albumin-based transport mechanism utilizing gp60/caveolae onmicrovascular endothelial cells (Desai et al., EORTC-NCI-AACR, 2004). Inaddition, both Cremophor® and Tween® 80 have been shown to stronglyinhibit the binding of paclitaxel to albumin, possibly affecting albuminbased transport (Desai et al., EORTC-NCI-AACR, 2004).

IDN5109 (Ortataxel) is a new taxane, currently in phase II, selected forits lack of cross-resistance in tumor cell lines expressing themultidrug resistant phenotype (MDR/Pgp) and inhibition of P-glycoprotein(Pgp) (Minderman; Cancer Chemother. Pharmacol. 2004; 53: 363-9). Due toits hydrophobicity, IDN5109 is currently formulated in the surfactantTweed® 80 (same vehicle as Taxotere®). Removal of surfactants fromtaxane formulations e.g., in the case of nanoparticle albumin-boundpaclitaxel (Abraxane®) showed improvements in safety and efficacy overtheir surfactant containing counterparts (O'Shaughnessy et al., SanAntonio Breast Cancer Symposium, Abstract #1122, December 2003). Tween®80 also strongly inhibited the binding of the taxane, paclitaxel, toalbumin, possibly compromising albumin based drug transport via the gp60receptor on microvessel endothelial cells (Desai et al., EORTC-NCI-AACR,2004).

The antitumor activity of colchicine, which is the major alkaloid of theautumn crocus, Colchicum autumnale, and the African climbing lily,Gloriosa superba, was first reported at the beginning of the 20^(th)century. The elucidation of its structure was finally completed fromX-ray studies and a number of total syntheses (see Shiau et al., J.Pharm. Sci. (1978) 67(3): 394-397). Colchicine is thought to be amitotic poison, particularly in tyhmic, intestinal, and hermatopoieticcells, which acts as a spindle poison and blocks the kinesis. Its effecton the mitotic spindle is thought to represent a special case of itseffects on various organized, labile, fibrillar systems concerned withstructure and movement.

Thiocolchicine dimer IDN5404 was selected for its activity in humanovarian sublime resistant to cisplatin and topotecan A2780-CIS andA2780-TOP. This effect was related to dual mechanisms of action, i.e.,microtubule activity as in Vinca alkaloids and a topoisomerase Iinhibitory effect different from camptothecin. (Raspaglio, BiochemicalPharmacology 69: 113-121 (2005)).

It has been found that nanoparticle compositions of a taxane (such asalbumin bound paclitaxel (Abraxane®)) have significantly lowertoxicities than other taxanes like Taxol® and Taxotere® withsignificantly improved outcomes in both safety and efficacy.

Combination chemotherapy, e.g., combining one or more chemotherapeuticagents or other modes of treatment, e.g., combining for example,chemotherapy with radiation or surgery and chemotherapy, have been foundto be more successful than single agent chemotherapeutics or individualmodes of treatment respectively.

Other references include U.S. Pub. No. 2006/0013819; U.S. Pub. No.2006/0003931; WO05/117986; WO05/117978; and WO05/000900.

More effective treatments for proliferative diseases, especially cancer,are needed.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of treating breast cancer basedon hormone receptor status (e.g., whether an individual's cell (e.g.,breast cancer cells) express or do not express the estrogen receptorand/or progesterone receptor) with nanoparticles comprising a taxane anda carrier protein (such as albumin)

The invention provides a method for treating breast cancer in anindividual, method includes: (a) determining hormone receptor status ofestrogen receptor and/or progesterone receptor; and (b) administering tothe individual an effective amount of a composition comprisingnanoparticles comprising a taxane and a carrier protein.

The invention also provides a method for treating breast cancer in anindividual, the method includes administering to the individual aneffective amount of a composition comprising nanoparticles comprising ataxane and a carrier protein, wherein hormone receptor status ofestrogen receptor and/or progesterone receptor is used as a basis forselecting the individual to receive treatment.

The invention provides a method of identifying an individual suitablefor breast cancer treatment, the method includes determining hormonereceptor status of estrogen receptor and/or progesterone receptor,wherein the individual is identified as suitable for breast cancertreatment with nanoparticles comprising a taxane and a carrier proteinif hormone receptor status is negative for both estrogen receptor andprogesterone receptor.

The invention further provides a method of assessing responsiveness ofan individual to a breast cancer therapy, the method includesdetermining hormone receptor status of estrogen receptor and/orprogesterone receptor, wherein the breast cancer therapy comprisesadministering a composition comprising nanoparticles comprising a taxaneand a carrier protein and wherein (a) the individual is likely moreresponsive to the therapy if hormone receptor status is negative forboth estrogen receptor and progesterone receptor and (b) the individualis likely less responsive to therapy if the hormone receptor status ispositive for estrogen receptor and/or progesterone receptor.

In some embodiments of any of the above methods, the hormone receptorstatus of estrogen receptor and/or progesterone receptor is determinedusing breast cancer tissue and/or cells. In some embodiments, thehormone receptor status of the individual is negative for both estrogenreceptor and progesterone receptor. In some embodiments, the breastcancer is locally advanced breast cancer. In some embodiments, thebreast cancer expresses HER2 (HER2+). In some embodiments, the breastcancer does not express HER2 (HER2−). In some embodiments, theindividual is human.

In some embodiments of any of the above methods, the method furtherincludes administering to the individual an effective amount of at leastone other chemotherapeutic agent. In some embodiments, the at least oneother chemotherapeutic agent comprises 5-fluorouracil, epirubicin, andcyclosphosphamide. In some embodiments, the composition comprisingnanoparticles (also referred to as “nanoparticle composition”) and thechemotherapeutic agent are administered simultaneously, either in thesame composition or in separate compositions. In some embodiments, thenanoparticle composition and the chemotherapeutic agent are administeredsequentially, i.e., the nanoparticle composition is administered eitherprior to or after the administration of the chemotherapeutic agent. Insome embodiments, the administration of the nanoparticle composition andthe chemotherapeutic agent are concurrent, i.e., the administrationperiod of the nanoparticle composition and that of the chemotherapeuticagent overlap with each other. In some embodiments, the administrationof the nanoparticle composition and the chemotherapeutic agent arenon-concurrent. For example, in some embodiments, the administration ofthe nanoparticle composition is terminated before the chemotherapeuticagent is administered. In some embodiments, the administration of thechemotherapeutic agent is terminated before the nanoparticle compositionis administered.

In some embodiments of any of the above methods, the method furtherincludes a second therapy including radiation therapy, surgery, orcombinations thereof. In some embodiments, the second therapy isradiation therapy. In some embodiments, the second therapy is surgery.In some embodiments, the first therapy is carried out prior to thesecond therapy. In some embodiments, the first therapy is carried outafter the second therapy.

In some embodiments of any of the above methods, the method furtherincludes metronomic therapy regimes. In some embodiments, there isprovided a method of administering a composition comprisingnanoparticles comprising a taxane and a carrier protein (such asalbumin), wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose oftaxane at each administration is about 0.25% to about 25% of its maximumtolerated dose following a traditional dosing regime. In someembodiments, there is provided a method of administering a compositioncomprising nanoparticles comprising paclitaxel and an albumin (such asAbraxane®), wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose ofpaclitaxel at each administration is about 0.25% to about 25% of itsmaximum tolerated dose following a traditional dosing regime. In someembodiments, the dose of the taxane (such as paclitaxel, for exampleAbraxane®) per administration is less than about any of 1%, 2%, 3%, 4%,5%, 6%, 7%. 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, 22%, 24%, or25% of the maximum tolerated dose. In some embodiments, the nanoparticlecomposition is administered at least about any of 1×, 2×, 3×, 4×, 5×,6×, 7× (i.e., daily) a week. In some embodiments, the intervals betweeneach administration are less than about any of 7 days, 6 days, 5 days, 4days, 3 days, 2 days, and 1 day. In some embodiments, the nanoparticlecomposition is administered over a period of at least about any of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36 months.

The methods of the invention generally comprise administration of acomposition comprising nanoparticles comprising a taxane and a carrierprotein. In some embodiments, the nanoparticle composition comprisesnanoparticles comprising paclitaxel and an albumin. In some embodiments,the paclitaxel albumin nanoparticles have an average diameter of nogreater than about 200 nm. In some embodiments, the paclitaxel/albuminnanoparticle composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some embodiments, the weight ratio ofthe albumin to paclitaxel in the composition is about 18:1 or less, suchas about 9:1 or less. In some embodiments, the paclitaxel is coated withalbumin. In some embodiments, the albumin is human serum albumin. Insome embodiments, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm and the paclitaxel/albumincomposition is substantially free (such as free) of surfactant (such asCremophor), In some embodiments, the paclitaxel/albumin nanoparticleshave an average diameter of no greater than about 200 nm and thepaclitaxel is coated with albumin. Other combinations of the abovecharacteristics are also contemplated. In some embodiments, thenanoparticle composition is Abraxane®. Nanoparticle compositionscomprising other taxanes (such as docetaxel and ortataxel) may alsocomprise one or more of the above characteristics. In some embodiments,the nanoparticles comprising a taxane and a carrier protein is thenanoparticle albumin bound paclitaxel, described, for example, in U.S.Pat. No. 6,566,405, and commercially available under the tradenameAbraxane®. In addition, the nanoparticles comprising a taxane and acarrier protein is considered to be nanoparticle albumin bound docetaxeldescribed for example in U.S. Patent Application Publication2005/0004002A1.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the taxane is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25 ng/m′ to about 25 mg/m². Insome embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®) and a carrier protein (such as albumin),wherein the paclitaxel is administered over a period of at least onemonth, wherein the interval between each administration is no more thanabout a week, and wherein the dose of the taxane at each administrationis about 0.25 mg/m² to about 25 mg/m². In some embodiments, the dose ofthe taxane (such as paclitaxel, for example Abraxane®) peradministration is less than about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 18, 20, 22, and 25 mg/m². In some embodiments, thenanoparticle composition is administered at least about any of 1×, 2×,3×, 4×, 5×, 6×, 7× (i.e., daily) a week. In some embodiments, theintervals between each administration are less than about any of 7 days,6 days, 5 days, 4 days, 3 days, 2 days, and 1 day. In some embodiments,the nanoparticle composition is administered over a period of at leastabout any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36months.

The invention also provides a kit comprising: (a) an agent for detectinghormone receptor status of estrogen receptor and/or progesteronereceptor of a breast cancer patient; and (b) a composition comprisingnanoparticles comprising a taxane and a carrier protein.

The invention further provides a kit comprising: (a) an agent fordetecting hormone receptor status of estrogen receptor and/orprogesterone receptor of a breast cancer patient; and (b) instructionsfor assessing likely responsiveness to therapy for treating breastcancer based on hormone receptor status of estrogen receptor and/orprogesterone receptor, wherein the therapy comprises administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein. In some embodiments, the instructions further provideinstructions for administering to the patient an effective amount of thecomposition.

In some embodiments of any of the kits, the hormone receptor status ofestrogen receptor and/or progesterone receptor is determined usingbreast cancer tissue and/or cells. In some embodiments, the hormonereceptor status of the individual is negative for both estrogen receptorand progesterone receptor. In some embodiments, the breast cancer islocally advanced breast cancer. In some embodiments, the breast cancerexpresses HER2 (HER2+). In some embodiments, the breast cancer does notexpress HER2 (HER2−). In some embodiments, the kit further includes atleast one other chemotherapeutic agent. In some embodiments, the atleast one other chemotherapeutic agent comprises 5-fluorouracil,epirubicin, and cyclosphosphamide.

In some embodiments, the kits comprise a composition comprisingnanoparticles comprising a taxane and a carrier protein. In someembodiments, the nanoparticle composition comprises nanoparticlescomprising paclitaxel and an albumin. In some embodiments, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm. In some embodiments, the paclitaxel/albuminnanoparticle composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some embodiments, the weight ratio ofthe albumin to paclitaxel in the composition is about 18:1 or less, suchas about 9:1 or less. In some embodiments, the paclitaxel is coated withalbumin. In some embodiments, the albumin is human serum albumin. Insome embodiments, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 run and the paclitaxel/albumincomposition is substantially free (such as free) of surfactant (such asCremophor). In some embodiments, the paclitaxel/albumin nanoparticleshave an average diameter of no greater than about 200 nm and thepaclitaxel is coated with albumin. Other combinations of the abovecharacteristics are also contemplated. In some embodiments, thenanoparticle composition is Abraxane®. Nanoparticle compositionscomprising other taxanes (such as docetaxel and ortataxel) may alsocomprise one or more of the above characteristics. In some embodiments,the nanoparticles comprising a taxane and a carrier protein is thenanoparticle albumin bound paclitaxel, described, for example, in U.S.Pat. No. 6,566,405, and commercially available under the tradenameAbraxane®. In addition, the nanoparticles comprising a taxane and acarrier protein is considered to be nanoparticle albumin bound docetaxeldescribed for example in U.S. Patent Application Publication2005/0004002A1.

These and other aspects and advantages of the present invention willbecome apparent from the subsequent detailed description and theappended claims. It is to be understood that one, some, or all of theproperties of the various embodiments described herein may be combinedto form other embodiments of the present invention.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows the effect of ABI-007 on rat aortic ring angiogenesis.FIG. 1B shows the effect of ABI-007 on human endothelial cellproliferation. FIG. 1C shows the effect of ABI-007 on endothelial celltube formation.

FIG. 2 shows the determination of an optimal biological dose of ABI-007for metronomic dosing. Shown are the levels of viable circulatingendothelial progenitors (CEPs) in peripheral blood of Balb/cJ mice inresponse to escalating doses of ABI-007. Untr′d, untreated control; S/A,saline/albumin vehicle control. Bars, mean±SE. * Significantly (p<0.05)different from the untreated control.

FIGS. 3A and 3B show the effects of ABI-007 and Taxol® used inmetronomic or MTD regimes on MDA-MB-231 (FIG. 3A) and PC3 (FIG. 3B)tumor growth tumor-bearing SCID mice. FIGS. 3C and 3D show the effectsof ABI-007 and Taxol® used in metronomic or MTD regimes on the bodyweight of MDA-MB-231 (FIG. 3C) and PC3 (FIG. 3D) tumor-bearing SCIDmice.

FIGS. 4A and 4B show changes in the levels of viable circulatingendothelial progenitors (CEPs) in peripheral blood of MDA-MB-231 (FIG.4A) and PC3 (FIG. 4B) tumor-bearing SCID mice after treatment with A,saline/albumin; B, Cremophor EL control; C, metronomic Taxol® 1.3 mg/kg;D, E, and F, metronomic ABI-007 3, 6, and 10 mg/kg, respectively; G, MTDTaxol®; H, MTD ABI-007. Bars, mean±SE. ^(a) Significantly (p<0.05)different from saline/albumin vehicle control. ^(b) Significantly(p<0.05) different from Cremophor EL vehicle control.

FIG. 5A shows intratumoral microvessel density of MDA-MB-231 (▪) and PC3(□) xenografts treated with A, saline/albumin; B, Cremophor EL control;C, metronomic Taxol® 1.3 mg/kg; D, E, and F, metronomic ABI-007 3, 6,and 10 mg/kg, respectively; G, MTD Taxol; H, MTD ABI-007. Bars, mean±SE.FIGS. 5B and 5C show the correlation between intratumoral microvesseldensity and the number of viable CEPs in peripheral blood in MDA-MB-231(FIG. 5B) and PC3 (FIG. 5C) tumor-bearing SCID mice.

FIG. 6 shows the effects of ABI-007 or Taxol used in metronomic or MTDregimes on basic fibroblast growth factor (bFGF)-induced angiogenesis inmatrigel plugs injected subcutaneously into the flanks of Balb/cJ mice.Treatments-A, saline/albumin; B, Cremophor EL control; C, metronomicTaxol 1.3 mg/kg; D, E, and F, metronomic ABI-007 3, 6, and 10 mg/kg,respectively; G, MTD Taxol; H, MTD ABI-007. Matrigel implanted withoutbFGF (−bFGF) served as negative control. Bars, mean±SE.

FIGS. 7A and 7B show necrosis in MDA-MB-231 tumor cells after treatmentwith saline control or Abraxane®. FIGS. 7C and 7D show hypoxia inMDA-MB-231 cells after treatment with saline control or Abraxane®.Arrows indicate sites of necrosis (FIGS. 7A and 7B) or sites of hypoxia(FIGS. 7C and 7D).

FIGS. 8A and 8B show the effect of VEGF-A and Avastin® onAbraxane®-treated cells in cytotoxicity and clonogenic assays. In FIG.8A, results are shown as viable cells as a percentage of untreatedcells. Dark circles indicate cells treated with Abraxane® alone; opencircles indicate cells treated with Abraxane® and VEGF-A; dark trianglesindicate cells treated with Abraxane® and Avastin®. In FIG. 8B, resultsare shown as the mean number of cell colonies per plate.

FIG. 9 shows the effect of Abraxane® and Avastin® treatment on thegrowth of MDA-MB-231 breast tumor xenografts. Dark squares indicate meantumor volume in saline-treated mice; dark circles indicate mean tumorvolume in Abraxane®-treated mice; dark diamonds indicate mean tumorvolume in Avastin®-treated mice; open diamonds indicate mean tumorvolume in Abraxane®+Avastin® (2 mg/kg)-treated mice; open circlesindicate mean tumor volume in Abraxane®+Avastin® (4 mg/kg)-treated mice;triangles indicate mean tumor volume in Abraxane®+Avastin® (8mg/kg)-treated mice. Two bars labeled ABX indicate the two Abraxane®treatment cycles.

FIGS. 10A and 10B show the effect of Abraxane® and Avastin® treatment onmetastasis of luciferase-expressing MDA-MB-231 tumor cells to the lymphnodes and lungs in tumor-bearing mice. Results are shown as levels ofluciferase activity in lymph node or lung cellular extracts.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating breast cancer basedon hormone receptor status (e.g., whether an individual's cell (e.g,breast cancer cells) express or do not express the estrogen receptorand/or progesterone receptor) with nanoparticles comprising a taxane anda carrier protein (such as albumin). The treatments can further involvecombination therapy comprising a first therapy comprising administrationof nanoparticles comprising a taxane and a carrier protein (such asalbumin) in conjunction with a second therapy such as radiation,surgery, administration of at least one other chemotherapeutic agent, orcombinations thereof for the treatment of breast cancer based on hormonestatus. The treatments can further involve metronomic therapy for thetreatment of breast cancer based on hormone receptor status.

The present invention involves the discovery that Abraxane®, due to itssuperior anti-tumor activity and reduced toxicity and side effects, canbe administered alone, in combination with other therapeutic drugsand/or treatment modalities and can also be used in metronomicchemotherapy to treat breast cancer based on hormone receptor status.Due to significantly improved safety profiles with compositionscomprising drug/carrier protein nanoparticles (such as Abraxane®), webelieve that monotherapy and combination chemotherapy with suchnanoparticle compositions (such as Abraxane®) is more effective thanmonotherapy with non-nanoparticle formulations or combinationchemotherapy with other drugs in treating breast cancer based on hormonereceptor status (e.g., not expressing the estrogen receptor and/orprogesterone receptor). In addition the use of nanoparticle composition(such as Abraxane®) in combination with radiation is also believed to bemore effective than combination of other agents with radiation in thetreatment of breast cancer based on hormone receptor status. Thus, thenanoparticle compositions (especially a paclitaxel/albumin nanoparticlecomposition, such as Abraxane®), when used alone, in combination withother chemotherapeutic agents, or when combined with other treatmentmodalities, should be very effective and overcome the deficiencies ofsurgery, radiation treatment, and chemotherapy in the treatment ofbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor).

The present invention in one its embodiments is the use of a compositioncomprising a taxane, such as Abraxane® for the treatment of breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor). In some embodiments,the present invention is the use of a first therapy comprising a taxane,such as Abraxane®, in combination with a second therapy such as anotherchemotherapeutic agent or agents, radiation, or the like for treatingbreast cancer based on hormone receptor status. The first therapycomprising a taxane and second therapy can be administered to a mammalhaving the proliferative sequentially, or they can be co-administered,and even administered simultaneously in the same pharmaceuticalcomposition.

Further, a metronomic dosing regime using Abraxane® has been found to bemore effective than the traditional MTD dosing schedule of the same drugcomposition. Such metronomic dosing regime of Abraxane® has also beenfound to be more effective than metronomic dosing of Taxol®.

The methods described herein are generally useful for treatment ofdiseases, particularly proliferative diseases. As used herein,“treatment” is an approach for obtaining beneficial or desired clinicalresults. For purposes of this invention, beneficial or desired clinicalresults include, but are not limited to, any one or more of: alleviationof one or more symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, preventing or delaying spread(e.g., metastasis) of disease, preventing or delaying occurrence orrecurrence of disease, delay or slowing of disease progression,amelioration of the disease state, and remission (whether partial ortotal). Also encompassed by “treatment” is a reduction of pathologicalconsequence of a proliferative disease. The methods of the inventioncontemplate any one or more of these aspects of treatment.

As used herein, a “proliferative disease” is defined as a tumor disease(including benign or cancerous) and/or any metastases, wherever thetumor or the metastasis are located, more especially a breast cancertumor. In some embodiments, the proliferative disease is cancer. In someembodiments, the proliferative disease is a benign or malignant tumor.Where hereinbefore and subsequently a tumor, a tumor disease, acarcinoma or a cancer are mentioned, also metastasis in the originalorgan or tissue and/or in any other location are implied alternativelyor in addition, whatever the location of the tumor and/or metastasis is.

The term “effective amount” used herein refers to an amount of acompound or composition sufficient to treat a specified disorder,condition or disease such as ameliorate, palliate, lessen, and/or delayone or more of its symptoms. In reference to cancers or other unwantedcell proliferation, an effective amount comprises an amount sufficientto cause a tumor to shrink and/or to decrease the growth rate of thetumor (such as to suppress tumor growth) or to prevent or delay otherunwanted cell proliferation. In some embodiments, an effective amount isan amount sufficient to delay development. In some embodiments, aneffective amount is an amount sufficient to prevent or delay occurrenceand/or recurrence. An effective amount can be administered in one ormore administrations. In the case of cancer, the effective amount of thedrug or composition may: (i) reduce the number of cancer cells; (ii)reduce tumor size; (iii) inhibit, retard, slow to some extent andpreferably stop cancer cell infiltration into peripheral organs; (iv)inhibit (i.e., slow to some extent and preferably stop) tumormetastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrenceand/or recurrence of tumor; and/or (vii) relieve to some extent one ormore of the symptoms associated with the cancer.

In some embodiments, there is provided a method of treating a primarytumor. In some embodiments, there is provided a method of treatingmetastatic cancer (that is, cancer that has metastasized from theprimary tumor). In some embodiments, there is provided a method oftreating cancer at advanced stage(s). In some embodiments, there isprovided a method of treating breast cancer (which may be HER2 positiveor HER2 negative), including, for example, advanced breast cancer, stageIV breast cancer, locally advanced breast cancer, and metastatic breastcancer. In some embodiments, there is provided a method of treatingsolid tumors (such as advanced solid tumors). In some embodiments, thereis provided a method of reducing cell proliferation and/or cellmigration. The present invention also provides methods of delayingdevelopment of any of the proliferative diseases described herein.

The term “individual” is a mammal, including humans. An individualincludes, but is not limited to, human, bovine, horse, feline, canine,rodent, or primate. In some embodiments, the individual is human. Theindividual (such as human) may have advanced disease or lesser extent ofdisease, such as low tumor burden. In some embodiments, the individualis at an early stage of a proliferative disease (such as cancer). Insome embodiments, the individual is at an advanced stage of aproliferative disease (such as an advanced cancer). In some embodiments,the individual is HER2 positive. In some embodiments, the individual isHER2 negative.

The methods may be practiced in an adjuvant setting. “Adjuvant setting”refers to a clinical setting in which an individual has had a history ofa proliferative disease, particularly cancer, and generally (but notnecessarily) been responsive to therapy, which includes, but is notlimited to, surgery (such as surgical resection), radiotherapy, andchemotherapy. However, because of their history of the proliferativedisease (such as cancer), these individuals are considered at risk ofdevelopment of the disease. Treatment or administration in the “adjuvantsetting” refers to a subsequent mode of treatment. The degree of risk(i.e., when an individual in the adjuvant setting is considered as “highrisk” or “low risk”) depends upon several factors, most usually theextent of disease when first treated. The methods provided, herein mayalso be practiced in a neoadjuvant setting, i.e., the method may becarried out before the primary/definitive therapy. In some embodiments,the individual has previously been treated. In some embodiments, theindividual has not previously been treated. In some embodiments, thetreatment is a first line therapy.

It is understood that aspect and embodiments of the invention describedherein include “consisting” and/or “consisting essentially of” aspectsand embodiments.

As is understood by one skilled in the art, reference to “about” a valueor parameter herein includes (and describes) embodiments that aredirected to that value or parameter per se. For example, descriptionreferring to “about X” includes description of “X”.

When hormone receptor status “is used as a basis” for administration ofthe treatment methods described herein, or selection for the treatmentmethods described herein, hormone receptor status is measured beforeand/or during treatment, and the values obtained are used by a clinicianin assessing any of the following: (a) probable or likely suitability ofan individual to initially receive treatment(s); (b) probable or likelyunsuitability of an individual to initially receive treatment(s); (c)responsiveness to treatment; (d) probable or likely suitability of anindividual to continue to receive treatment(s); (e) probable or likelyunsuitability of an individual to continue to receive treatment(s); (f)adjusting dosage; or (g) predicting likelihood of clinical benefits.

Method of Treatment

The present invention provides methods of treating breast cancer basedon hormone receptor status of the breast cancer tissue with acomposition comprising nanoparticles comprising a taxane and a carrierprotein. In some embodiments, the hormone receptor status is determinedbased on the expression of a hormone receptor such as the estrogenreceptor (ER) or the progesterone receptor (PgR). In some embodiments,the hormone receptor status is determined based on the expression of ahormone receptor such as the estrogen receptor and the progesteronereceptor of the breast cancer tissue and/or cells.

In some embodiments, the hormone receptor status is low for one or morehormone receptors such as the estrogen receptor and/or the progesteronereceptor. In some embodiments, the individual is likely more responsiveto the therapy if hormone receptor status is low for both estrogenreceptor and progesterone receptor. In some embodiments, the hormonereceptor status does not express (i.e., is negative for) one or morehormone receptors such as the estrogen receptor (ER) or the progesteronereceptor (PgR). In some embodiments, the hormone receptor status of thebreast cancer tissue does not express is negative for) both the estrogenreceptor (ER) and the progesterone receptor (PgR). In some embodiments,the individual is likely more responsive to the therapy if hormonereceptor status is negative for both estrogen receptor and progesteronereceptor. In some embodiments, the individual expresses (i.e., ispositive for) either the estrogen receptor or the progesterone receptor.In some embodiments, the individual expresses (i.e., is positive for)both the estrogen receptor and the progesterone receptor. In someembodiments, the individual is likely less responsive to therapy if thehormone receptor status is positive for the estrogen receptor and/or theprogesterone receptor.

In some embodiments, the breast cancer further expresses HER2 (HER2+).In some embodiments, the breast cancer further does not express HER2(HER2−).

In some embodiments, the nanoparticles have an average diameter of nogreater than about 200 nm. In some embodiments, the nanoparticlecomposition is substantially free (such as free) of surfactant (such asCremophor). In some embodiments, the taxane is paclitaxel. In someembodiments, the carrier protein is albumin. In some embodiments, thealbumin is human serum albumin. In some embodiments, the weight ratio ofthe albumin to paclitaxel in the composition is about 18:1 or less, suchas about 9:1 or less. In some embodiments, the paclitaxel is coated withalbumin. In some embodiments, the paclitaxel/albumin nanoparticles havean average diameter of no greater than about 200 nm and thepaclitaxel/albumin composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some embodiments, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel is coated with albumin. In someembodiments, the nanoparticle composition is Abraxane®.

Estrogen, mediated through the estrogen receptor (ER), plays a majorrole in regulating the growth and differentiation of normal breastepithelium (Pike M C et al. Epidemiologic Reviews (1993) 15(1): 17-35;Henderson B E et al. Cancer Res. (1988) 48: 246-253). It stimulates cellproliferation and regulates the expression of other genes, including theprogesterone receptor (PgR). PgR then mediates the mitogenic effect ofprogesterone, further stimulating proliferation (Pike et al., 1993;Henderson et al., 1988). The molecular differences between estrogenreceptor (“ER”) negative and ER positive tumors are significant in lightof clinical observations which indicate that the nature and biologicalbehavior of ER positive and ER negative tumors are distinct even in theabsence of hormonal therapy. For example, ER negative cancers tend torecur sooner and show a different rate of recurrence in distant organsites compared to ER positive tumors. Clinical observations andmolecular profiling data suggest that tumors not expressing both ER andPgR represent a different clinical entity in terms of chemotherapyresponsiveness. (Colleoni et al., Annals of Oncology 11(8): 1057(2000)). Thus, ER negative and ER positive breast cancers are twodistinct disease entities rather than phenotypic variations of the samedisease.

In some embodiments, the method comprises identifying a breast cancerpatient based on a hormone receptor status of patients having tumortissue not expressing both ER and PgR. Suitable patients areadministered an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel, docetaxel, orortataxel) and a carrier protein (such as albumin). In some embodiments,the method further comprises administering to the patient an effectiveamount of at least one other chemotherapeutic agent. The at least oneother chemotherapeutic agent may be administered concurrently orsequentially with the taxane nanoparticles. In some embodiments the atleast one other chemotherapeutic agent comprises 5-Fluoruracil,Epirubicin and Cyclophosphamide (FEC) administered concurrently orsequentially. These methods have higher efficacy in ER(−)/PgR(−)populations in all patient populations, both HER-2 positive and HER-2negative.

Various methods can be used to determine hormone receptor status andmeasure hormone receptor levels (e.g., estrogen receptor levels and/orprogesterone receptor levels) in a sample (e.g., breast cancer tissue).Methods for measuring RNA levels include, without limitation,hybridization (e.g., Northern blotting of separated RNAs, microarray,and dot or slot blotting or total RNA) and PCR-based methods (e.g.,RT-PCR and quantitative real-time PCR). For example, hybridization canbe done by Northern analysis to identify an RNA sequence that hybridizesto a probe. The probe can be labeled with a radioisotope such as ³²P, anenzyme, digoxygenin, or by biotinylation. The RNA to be analyzed can beelectrophoretically separated on an agarose or polyacrylamide gel,transferred to nitrocellulose, nylon, or other suitable membrane, andhybridized with the probe using standard techniques well known in theart such as those described in sections 7.39-7.52 of Sambrook et al.,(1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory,Plainview, N.Y.

As standard Northern blot assays can be used to ascertain the level of aparticular RNA in a sample from a mammal, so can PCR-based methods suchas quantitative real-time PCR. In one embodiment, reverse transcriptionusing random hexamer oligonucleotide primers can be performed on totalmRNA isolated from a cancer sample. The resulting cDNA then can be usedas template in quantitative real-time PCR experiments using forward andreverse oligonucleotide primers in the presence of a specific probe(e.g., a probe having a 5′ fluorescent reporter dye at one end and a 3′quencher dye at the other end). Reactions can be monitored using thepoint during cycling when amplification of a PCR product is firstdetected, rather than the amount of PCR product accumulated after afixed number of cycles. The resulting quantitated PCR product levels canbe correlated to the mRNA levels in the original cancer sample, and themRNA levels can in turn be correlated with the aggressiveness of thatcancer.

Methods for measuring polypeptide hormone receptor levels (e.g.,estrogen receptor protein levels and/or progesterone receptor proteinlevels) include, without limitation, ELISA-, immunohistochemistry-, andimmunofluorescence-based techniques. Such methods typically employantibodies having specific binding affinity for a particularpolypeptide. “Specific binding affinity” refers to an antibody's abilityto interact specifically with a particular polypeptide withoutsignificantly cross-reacting with other different polypeptides in thesame environment. An antibody having specific binding affinity forestrogen receptor or progesterone receptor can interact with estrogenreceptor or progesterone receptor polypeptides.

Estrogen receptor or progesterone receptor polypeptide levels in abreast cancer sample can, for example, be measured using a quantitativesandwich ELISA technique. Breast cancer tissue samples can behomogenized and extracted, and aliquots of the extracts added toseparate wells of a microtiter plate pre-coated with antibodies specificfor estrogen receptor or progesterone receptor. After protein bindingand subsequent washing, enzyme-linked antibodies specific for estrogenreceptor or progesterone receptor can be added to the wells. Afterantibody binding and subsequent washing, a substrate solution containinga label-conjugated IgG can be added to the wells (e.g., horseradishperoxidase (HRP)-conjugated IgG). The label then can be quantitated byspectrophotometry and the quantitated levels compared to a control levelor baseline. The resulting quantitated polypeptide levels can becorrelated with the aggressiveness of that cancer.

Polypeptide levels also can be measured by immunohistochemistry. Forexample, a section of a breast cancer tissue sample can be treated withanti-estrogen receptor or anti-progesterone receptor antibodies.Negative control sections can be incubated with pre-immune rabbit ormouse serum in lieu of primary antibodies. After antibody binding andsubsequent washing, the primary antibodies can be detected withappropriate label-conjugated secondary antibodies (e.g, gold-conjugatedor enzyme-conjugated antibodies). The label is then developed andquantitated using an image analysis system. The resulting quantitatedpolypeptide levels can be correlated with the aggressiveness of thatcancer. Although samples can be processed individually, samples fromdifferent tissues or from a population of different patients can beprocessed simultaneously. Such processing methods include, withoutlimitation, tissue microarrays, as described by Kononen et al., (1998)Nature Med. 4: 844-847.

Suitable antibodies for ELISA-, immunohistochemistry- andimmunofluorescence-based methods can be obtained using standardtechniques. In addition, commercially available antibodies topolypeptides associated with cancer aggressiveness can be used.

Based upon the various methods of measuring described above and known inthe art, the hormone receptor status such as progesterone receptor andestrogen receptor may be determined. For example, usingimmunohistochemistry and measuring nuclear reactivity of estrogenreceptor and/or the progesterone receptor, the percentage ofimmunoreactive cells expressing estrogen receptor and/or theprogesterone receptor recorded as the percentage of immunoreactive cellsover at least 2,000 neoplastic cells may be determined. Breast cancertissue is characterized as having a low hormone receptor status ifgreater than or equal to about 1% to about 10% of the cells of thebreast cancer tissue express the estrogen receptor and/or theprogesterone receptor. Breast cancer tissues is characterized as notexpressing or negative for a hormone receptor such as estrogen receptorand/or the progesterone receptor if less than about 1% of the cells ofthe breast cancer tissue express the hormone receptor. In someembodiments, less than about any of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, or 0.9% of the cells of the breast cancer tissue express ahormone receptor such as estrogen receptor or the progesterone receptor.In some embodiments, less than about any of 0.1%, 0.2%, 0.3%, 0.4%,0.5%, 0.6%, 0.7%, 0.8%, or 0.9% of the cells of the breast cancer tissueexpress the estrogen receptor and the progesterone receptor. In someembodiments, 0% of the cells of the breast cancer tissue express ahormone receptor such as estrogen receptor or the progesterone receptor.In some embodiments, 0% of the cells of the breast cancer tissue expressthe estrogen receptor and the progesterone receptor.

Combination Therapy with Chemotherapeutic Agent

The present invention provides methods of treating breast cancer basedon hormone receptor status (e.g., not expressing the estrogen receptorand/or progesterone receptor) in an individual, comprising administeringto the individual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane and a carrier protein (such asalbumin); and b) an effective amount of at least one otherchemotherapeutic agent. In some embodiments, the taxane is any of (andin come embodiments consisting essentially of) paclitaxel, docetaxel,and ortataxel. In some embodiments, the nanoparticle compositioncomprises Abraxane®. In some embodiments, the chemotherapeutic agent isany of (and in some embodiments selected from the group consisting of)antimetabolite agents (including nucleoside analogs), platinum-basedagents, alkylating agents, tyrosine kinase inhibitors, anthracyclineantibiotics, vinca alkloids, proteasonle inhibitors, macrolides, andtopoisomerase inhibitors.

In some embodiments, the hormone receptor status is low for one or morehormone receptors such as the estrogen receptor or the progesteronereceptor. In some embodiments, the individual is likely more responsiveto the therapy if hormone receptor status is low for both estrogenreceptor and progesterone receptor. In some embodiments, the hormonereceptor status does not express (i.e., is negative for) one or morehormone receptors such as the estrogen receptor (ER) or the progesteronereceptor (PgR). In some embodiments, the hormone receptor status of thebreast cancer tissue does not express (i.e., is negative for) both theestrogen receptor (ER) and the progesterone receptor (PgR). In someembodiments, the individual is likely more responsive to the therapy ifhormone receptor status is negative for both estrogen receptor andprogesterone receptor. In some embodiments, the individual expresses(i.e., is positive for) either the estrogen receptor or the progesteronereceptor. In some embodiments, the individual expresses (i.e., ispositive for) both the estrogen receptor and the progesterone receptor.In some embodiments, the individual is likely less responsive to therapyif the hormone receptor status is positive for the estrogen receptorand/or the progesterone receptor.

In some embodiments, the breast cancer tissue further expresses HER2(HER2+). In some embodiments, the breast cancer tissue further does notexpress HER2 (HER2−).

In some embodiments, the method comprises administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin; and b) an effectiveamount of at least one other chemotherapeutic agent. In someembodiments, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm. In some embodiments, thepaclitaxel; albumin nanoparticle composition is substantially free (suchas free) of surfactant (such as Cremophor). In some embodiments, theweight ratio of the albumin to paclitaxel in the composition is about18:1 or less, such as about 9:1 or less. In some embodiments, thepaclitaxel is coated with albumin. In some embodiments, the paclitaxel;albumin. nanoparticles have an average diameter of no greater than about200 nm and the paclitaxel/albumin composition is substantially free(such as free) of surfactant (such as Cremophor). In some embodiments,the paclitaxel/albumin nanoparticles have an average diameter of nogreater than about 200 nm and the paclitaxel is coated with albumin. Insome embodiments, the nanoparticle composition is Abraxane®.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individualcomprising administering to the individual a) an effective amount ofAbraxane®, and b) an effective amount of at least one otherchemotherapeutic agent. Preferred drug combinations for sequential orco-administration or simultaneous administration with Abraxane® arethose which show enhanced antiproliferative activity when compared withthe single components alone, especially combinations that that lead toregression of proliferative tissues and/or cure from proliferativediseases.

The chemotherapeutic agents described herein can be the agentsthemselves, pharmaceutically acceptable salts thereof, andpharmaceutically acceptable esters thereof, as well as stereoisomers,enantiomers, racemic mixtures, and the like. The chemotherapeutic agentor agents as described can be administered as well as a pharmaceuticalcomposition containing the agent(s), wherein the pharmaceuticalcomposition comprises a pharmaceutically acceptable carrier vehicle, orthe like.

The chemotherapeutic agent may be present in a nanoparticle composition.For example, in some embodiments, there is provided a method of treatingbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual: a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin); and b) an effective amount of a compositioncomprising nanoparticles comprising at least one other chemotherapeuticagent and a carrier protein (such as albumin). In some embodiments, themethod comprises administering to the individual: a) an effective amountof a composition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®); and b) an effective amount of a compositioncomprising nanoparticles comprising at least one other chemotherapeuticagent and a carrier protein (such as albumin). In some embodiments, thechemotherapeutic agent is any of (and in some embodiments selected fromthe group consisting of) thiocoichicine or its derivatives (such asdimeric thiocolehicine, including for example nab-5404, nab-5800, andnab-5801), rapamycin or its derivatives, and geldanamycin or itsderivatives (such as 17-allyl amino geldanamycin (17-AAG)). In someembodiments, the chemotherapeutic agent is rapamycin. In someembodiments, the chemotherapeutic agent is 17-AAG.

An exemplary and non-limiting list of chemotherapeutic agentscontemplated is provided herein. Suitable chemotherapeutic agentsinclude, for example, vinca alkaloids, agents that disrupt microtubuleformation (such as colchicines and its derivatives), anti-angiogenicagents, therapeutic antibodies, EGFR targeting agents, tyrosine kinasetargeting agent (such as tyrosine kinase inhibitors), transitional metalcomplexes, proteasome inhibitors, antimetabolites (such as nucleosideanalogs), alkylating agents, platinum-based agents, anthracyclineantibiotics, topoisomerase inhibitors, macrolides, therapeuticantibodies, retinoids (such as all-trans retinoic acids or a derivativesthereof); geldanamycin or a derivative thereof (such as 17-AAG), andother standard chemotherapeutic agents well recognized in the art.

In some embodiments, the chemotherapeutic agent is any of (and in someembodiments selected from the group consisting of) adriamycin,colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin,doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone,fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin,etoposide, interferons, carnptothecin and derivatives thereof,phenesterine, taxanes and derivatives thereof (e.g., paclitaxel andderivatives thereof, taxotere and derivatives thereof, and the like),topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404,nab-5800, nab-5801, Irinotecan, HKP, Ortataxel, gemcitabine, Herceptin®,vinorelbine, Doxil®, capecitabine, Alimta®, Avastin®, Velcade®,Tarceva®, Neulasta®, Lapatinib, Sorafenib, derivatives thereof,chemotherapeutic agents known in the art, and the like. In someembodiments, the chemotherapeutic agent is a composition comprisingnanoparticles comprising a thiocolchicine derivative and a carrierprotein (such as albumin).

In some embodiments, the chemotherapeutic agent is a antineoplasticagent including, but is not limited to, carboplatin, Navelbine®(vinorelbine), anthracycline (Doxil®), lapatinib (GW57016), Herceptin®,gemcitabine (Gemzar®), capecitabine (Xeloda®), Alimta®, cisplatin,5-fluorouracil, epirubicin, cyclophosphamide, Avastin®, Velcade®, etc.

In some embodiments, the chemotherapeutic agent is an antagonist ofother factors that are involved in tumor growth, such as EGFR, ErbB2(also known as Herb), ErbB3, ErbB4, or TNF. Sometimes, it may bebeneficial to also administer one or more cytokines to the individual.In some embodiments, the therapeutic agent is a growth inhibitory agent.Suitable dosages for the growth inhibitory agent are those presentlyused and may be lowered due to the combined action (synergy) of thegrowth inhibitory agent and the taxane.

In some embodiments, the chemotherapeutic agent is a chemotherapeuticagent other than an anti-VEGF antibody, a HER2 antibody, interferon, andan HGFβ antagonist.

Reference to a chemotherapeutic agent herein applies to thechemotherapeutic agent or its derivatives and accordingly the inventioncontemplates and includes either of these embodiments (agent; agent orderivative(s)). “Derivatives” or “analogs” of a chemotherapeutic agentor other chemical moiety include, but are not limited to, compounds thatare structurally similar to the chemotherapeutic agent or moiety or arein the same general chemical class as the chemotherapeutic agent ormoiety. In some embodiments, the derivative or analog of thechemotherapeutic agent or moiety retains similar chemical and/orphysical property (including, for example, functionality) of thechemotherapeutic agent or moiety.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of a tyrosinekinase inhibitor. In some embodiments, the invention provides a methodof treating breast cancer based on hormone receptor status (e.g., notexpressing the estrogen receptor and/or progesterone receptor) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®), and b) an effective amount of atyrosine kinase inhibitor. Suitable tyrosine kinase inhibitors include,for example, imatinib (Gleevec®), gefitinib (Iressa®), Tarceva, Sutent®(sunitinib malate), and Lapatinib. In some embodiments, the tyrosinekinase inhibitor is lapatinib. In some embodiments, the tyrosine kinaseinhibitor is Tarceva. Tarceva is a small molecule human epidermal growthfactor type 1/epidermal growth factor receptor (HER1/EGFR) inhibitorwhich demonstrated, in a Phase III clinical trial, an increased survivalin advanced non-small cell lung cancer (NSCLC) individuals. In someembodiments, the method is for treatment of breast cancer, includingtreatment of metastatic breast cancer and treatment of breast cancer ina neoadjuvant setting. In some embodiments, the method is for treatmentof advanced solid tumor. In some embodiments, there is provided a methodto inhibit the proliferation of EGFR expressing tumors in a mammalcomprising administering to a mammal infected with such tumors Abraxane®and gefitinib, wherein the gefitinib is administered by pulse-dosing.

In some embodiments, the invention provides a method of treating breastcancer based on honuone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of anantimetabolite agent (such as a nucleoside analog, including for examplepurine analogs and pyrimidine analogs). In some embodiments, theinvention provides a method of treating breast cancer based on hormonereceptor status (e.g., not expressing the estrogen receptor and/orprogesterone receptor) in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),and b) an effective amount of an antimetabolite agent. An “antimetabolicagent” is an agent which is structurally similar to a metabolite, butcannot be used by the body in a productive manner, Many antimetaboliteagents interfere with production of nucleic acids, RNA and DNA. Forexample, the antimetabolite can be a nucleoside analog, which includes,but is not limited to, azacitidine, azathioprine, capecitabine(Xeloda®), cytarabine, cladribine, cytosine arabinoside (ara-C,cytosar), doxifluridine, fluorouracil (such as 5-fluorouracil), UFT,hydoxyurea, gemcitabine, mercaptopurine, methotrexate, thioguanine (suchas 6-thioguanine). Other anti-metabolites include, for example,L-asparaginase (ELspa), decarbazine (DTIC), 2-deoxy-D-glucose, andprocarbazine (matulane). In some embodiments, the nucleoside analog isany of (and in some embodiments selected from the group consisting of)gemcitabine, fluorouracil, and capecitabine. In some embodiments, themethod is for treatment of metastatic breast cancer or locally advancedbreast cancer. In some embodiments, the method is for first linetreatment of metastatic breast cancer. In some embodiments, the methodis for treatment of breast cancer in a neoadjuvant setting.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of an alkylatingagent. In some embodiments, the invention provides a method of treatingbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®), and b) an effective amount of an alkylatingagent. Suitable alkylating agents include, but are not limited to,cyclophosphamide (Cytoxan), mechlorethamine, chlorambucil, melphalan,carmustine (BCNU), thiotepa, busulfan, alkyl sulphonates, ethyleneimines, nitrogen mustard analogs, estramustine sodium phosphate,ifosfamide, nitrosoureas, lomustine, and streptozocin. In someembodiments, the alkylating agent is cyclophosphamide. In someembodiments, the cyclophosphamide is administered prior to theadministration of the nanoparticle composition. In some embodiments, themethod is for treatment of an early stage breast cancer. In someembodiments, the method is for treatment of a breast cancer in anadjuvant or a neoadjuvant setting.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of aplatinum-based agent. In some embodiments, the invention provides amethod of treating breast cancer based on hormone receptor status (e.g.,not expressing the estrogen receptor and/or progesterone receptor) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®), and b) an effective amount of aplatinum-based agent. Suitable platinum-based agents include, but arenot limited to, carboplatin, cisplatin, and oxaliplatin. In someembodiments, the platinum-based agent is carboplatin. In someembodiments, the method is for treatment of breast cancer (HER2 positiveor HER2 negative, including metastatic breast cancer and advanced breastcancer).

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of ananthracycline antibiotic. In some embodiments, the invention provides amethod of treating a proliferative disease (such as cancer) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®) and a carrier protein (such asalbumin), and b) an effective amount of an anthracycline antibiotic.Suitable anthracycline antibiotic include, but are not limited to,Doxil®, actinomycin, dactinomycin, daunorubicin (daunomycin),doxorubicin (adriamycin), epirubicin, idarubicin, mitoxantrone,valrubicin. In some embodiments, the anthracycline is any of (and insome embodiments selected from the group consisting of) Doxil®,epirubicin, and doxorubicin. In some embodiments, the method is fortreatment of an early stage breast cancer. In some embodiments, themethod is for treatment of a breast cancer in an adjuvant or aneoadjuvant setting.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of a vincaalkaloid. In some embodiments, the invention provides a method oftreating breast cancer based on hormone receptor status (e.g., notexpressing the estrogen receptor and/or progesterone receptor) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®) and a carrier protein (such asalbumin), and b) an effective amount of a vinca alkaloid. Suitable vincaalkaloids include, for example, vinblastine, vincristine, vindesine,vinorelbine (Navelbine®), and VP-16. In some embodiments, the vincaalkaloid is vinorelbine (Navelbine®). In some embodiments, the method isfor treatment of stage IV breast cancer.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of a macrolide. Insome embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®) and a carrier protein (such as albumin), andb) an effective amount of a macrolide. Suitable macrolides include, forexample, rapamycin, carbomycin, and erythromycin. In some embodiments,the macrolide is rapamycin or a derivative thereof. In some embodiments,the method is for treatment of a solid tumor.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of a topoisomeraseinhibitor. In some embodiments, the invention provides a method oftreating breast cancer based on hormone receptor status (e.g., notexpressing the estrogen receptor and/or progesterone receptor) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®) and a carrier protein (such asalbumin), and b) an effective amount of a topoisomerase inhibitor. Insome embodiments, the chemotherapeutic agent is a topoisomeraseinhibitor, including, for example, inhibitor of topoisomerase I andtopoisomerase II. Exemplary inhibitors of topoisomerase I include, butare not limited to, camptothecin, such as irinotecan and topotecan.Exemplary inhibitors of topoisomerase II include, but are not limitedto, amsacrine, etoposide, etoposide phosphate, and teniposide.

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of anantiangiogenic agent. In some embodiments, the invention provides amethod of treating breast cancer based on hormone receptor status (e.g.,not expressing the estrogen receptor and/or progesterone receptor) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®) and a carrier protein (such asalbumin), and b) an effective amount of an antiangiogenic agent. In someembodiments, the method is for treatment of metastatic breast cancer andbreast cancer in an adjuvant setting or a neoadjuvant setting.

Many anti-angiogenic agents have been identified and are known in theart, including those listed by Carmeliet and Jain (2000). Theanti-angiogenic agent can be naturally occurring or non-naturallyoccurring. In some embodiments, the chemotherapeutic agent is asynthetic antiangiogenic peptide. For example, it has been previouslyreported that the antiangiogenic activity of small synthetic pro-apopticpeptides comprise two functional domains, one targeting the CD13receptors (aminopeptidase N) on tumor microvessels and the otherdisrupting the mitochondrial membrane following internalization. Nat Med(1999) 5(9): 1032-8. A second generation dimeric peptide,CNGRC-GG-d(KLAKLAK)2, named HKP (Hunter Killer Peptide) was found tohave improved antitumor activity. Accordingly, in some embodiments, theantiangiogenic peptide is HKP. In some embodiments, the antiangiogenicagent is other than an anti-VEGF antibody (such as Avastin®).

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of a proteasomeinhibitor, such as bortezomib (Velcade). In some embodiments, theinvention provides a method of treating breast cancer based on hormonereceptor status (e.g., not expressing the estrogen receptor and/orprogesterone receptor) in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®)and a carrier protein (such as albumin), and b) an effective amount of aproteasome inhibitor such as bortezomib (Velcade®).

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), and b) an effective amount of a therapeuticantibody. In some embodiments, the invention provides a method oftreating breast cancer based on hormone receptor status (e.g., notexpressing the estrogen receptor and/or progesterone receptor) in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®) and a carrier protein (such asalbumin), and b) an effective amount of a therapeutic antibody. Suitabletherapeutic antibodies include, but are not limited to, anti-VEGFantibody (such as Avastin® (bevacizumab)), anti-HER2 antibody (such asHerceptin® (trastuzumab)), Erbitux® (cetuximab), Campath (alemtuzumab),Myelotarg (gemtuzumab), Zevalin (ibritumomab tiuextan, Rituxan(rituximab), and Bexxar (tositumomab). In some embodiments, thechemotherapeutic agent is Erbitux® (cetuximab). In some embodiments, thechemotherapeutic agent is a therapeutic antibody other than an antibodyagainst VEGF or HER2. In some embodiments, the method is for treatmentof HER2 positive breast cancer, including treatment of advanced breastcancer, treatment of metastatic cancer, treatment of breast cancer in anadjuvant setting, and treatment of breast cancer in a neoadjuvantsetting. In some embodiments, the method is for treatment of any ofmetastatic breast cancer and breast cancer in an adjuvant setting or aneoadjuvant setting. For example, in some embodiments, there is provideda method for treatment of HER2 positive metastatic breast cancer in anindividual, comprising administering to the individual 125 mg/m²paclitaxel/albumin nanoparticle composition (such as Abraxane®) weeklyfor three weeks with the fourth week off, concurrent with theadministration of Herceptin®.

In some embodiments, there is provided a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual: a) an effective amount of acomposition comprising nanoparticles comprising taxane and a carrierprotein, and b) an effective amount of an anti-VEGF antibody. In someembodiments, the effective amounts of the taxane nanoparticlecomposition and the anti-VEGF antibody synergistically inhibit cellproliferation (such as tumor cell growth). In some embodiments, at leastabout 10% (including for example at least about any of 20%, 30%, 40%,60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited. In someembodiments, the taxane is paclitaxel. In some embodiments, theanti-VEGF antibody is bevacizumab (such as Avastin®). In someembodiments, the taxane is paclitaxel and the anti-VEGF antibody isbevacizumab (such as Avastin®). In some embodiments, the taxane in thenanoparticle in the composition is administered by intravenousadministration. In some embodiments, the anti-VEGF antibody isadministered by intravenous administration. In some embodiments, boththe taxane in the nanoparticle composition and the anti-VEGF antibodyare administered by intravenous administration.

In some embodiments, there is provided a method of inhibiting breastcancer tumor metastasis based on hormone receptor status (e.g., notexpressing the estrogen receptor and/or progesterone receptor) in anindividual, comprising administering to the individual: a) an effectiveamount of a composition comprising nanoparticles comprising taxane and acarrier protein, and b) an effective amount of an anti-VEGF antibody. Insome embodiments, the effective amounts of the taxane nanoparticlecomposition and the anti-VEGF antibody synergistically inhibit tumormetastasis. In some embodiments, at least about 10% (including forexample at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or100%) metastasis is inhibited. In some embodiments, method of inhibitingmetastasis to lymph node is provided. In some embodiments, method ofinhibiting metastasis to the lung is provided. In some embodiments, thetaxane is paclitaxel. In some embodiments, the anti-VEGF antibody isbevacizumab (such as Avastin®). In some embodiments, the taxane ispaclitaxel and the anti-VEGF antibody is bevacizumab (such as Avastin®).In some embodiments, the taxane in the nanoparticle in the compositionis administered by intravenous administration. In some embodiments, theanti-VEGF antibody is administered by intravenous administration. Insome embodiments, both the taxane in the nanoparticle composition andthe anti-VEGF antibody are administered by intravenous administration.

Suitable dosages for anti-VEGF antibody include, for example, about 1mg/kg to about 20 mg/kg, including for example about 1 mg/kg to about 15mg/kg (such as about any of 2, 4, 6, 8, 10, or 12 mg/kg). In someembodiments, the dosage of the anti-VEGF antibody is about 40 mg/m² toabout 600 mg/m², including for example about 100 mg/m² to about 400mg/m² (such as about any of 100, 200, or 300 mg/m²). In someembodiments, the anti-V EGF antibody is bevacizumab (such as Avastin®).

Suitable combinations of the amounts of taxane in a nanoparticlecomposition and the anti-VEGF antibody include, for example, about 1mg/kg to about 20 mg/kg (such as about any of 2, 5, 10, or 15 mg/kg)taxane in a nanoparticle composition and about 1 mg/kg to about 20 mg/kg(such as about any of 2, 4, 6, 8, 10, 12, 14, 16, or 18 mg/kg) anti-VEGFantibody; about 3 mg/m² to about 400 mg/m² (such as about any of 6, 10,15, 30, 45, 60, 100, 150, 200, or 300 mg/m²) taxane in a nanoparticlecomposition and 40 mg/m² to about 600 mg/m², including for example about100 mg/m² to about 400 mg/m² (such as about any of 100, 200, or 300mg/m²) anti-VEGF antibody; about 3 mg/m² to about 300 mg/m² (such asabout any of 6, 10, 15, 30, 45, 60, 100, 150, 200, or 300 mg/m²) taxanein a nanoparticle composition and about 1 mg/kg to about 20 mg/kg (suchas about any of 2, 4, 6, 8, 10, 12, 14, 16, or 18 mg/kg) anti-VEGFantibody. In some embodiments, the method comprises administering to anindividual at least about 200 mg/m² taxane in a nanoparticle compositionand at least about any of 2, 4, 8, or 10 mg/kg anti-VEGF antibody.

In some embodiments of the method, the taxane nanoparticle compositionand the anti-VEGF antibody are administered simultaneously to theindividual. In some embodiments of the method, the administration of thenanoparticle composition and the chemotherapeutic agent are concurrent.One exemplary dosing regime for the combination therapy of taxanenanoparticle composition includes administration of 100 mg/m2-300 mg/m²(such as 200 mg/m²) taxane in nanoparticle composition at least weekly(including for example every 1, 2, 3, 4, 5, or 6 days) concurrent withadministration of 2 mg/kg-15 mg/kg (such as any of 4, 6, 8, 10 mg/kg or15 mg/kg) anti-VEGF antibody every two weeks or more frequently (forexample every week, twice every week, or three times a week).

In some embodiments, the taxane nanoparticle composition and theanti-VEGF antibody are administered sequentially to the individual. Forexample, in some embodiments, the taxane nanoparticle composition isadministered for at least one (such as at least any of two, three, four,five, or six) cycles prior to the administration of the anti-VEGFantibody. This is then followed by the administration of an anti-VEGFantibody for at least once (such as twice) a week for at least about 3(such as 4, 5, or 6) weeks. One exemplary dosing regime for thecombination therapy of taxane nanoparticle composition (such aspaclitaxel/albumin nanoparticle composition, for example Abraxane®) andanti-VEGF antibody (such as bevacizumab, for example Avastin®) includesadministration of 10 mg/kg taxane in a nanoparticle composition dailyfor 5 days in two cycles separated by one week followed byadministration of an anti-VEGF antibody at dosages of 2 mg/kg, 4 mg/kg,or 8 mg/kg twice a week for 6 weeks.

In some embodiments, two or more chemotherapeutic agents areadministered in addition to the taxane in the nanoparticle composition.These two or more chemotherapeutic agents may (but not necessarily)belong to different classes of chemotherapeutic agents. Examples ofthese combinations are provided herein. Other combinations are alsocontemplated.

In some embodiments, there is provided a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), b) an effective amount of an antimetabolite(such as a nucleoside analog, for example, gemcitabine), and c) ananthracycline antibiotic (such as epirubicin). In some embodiments,there is provided a method of treating breast cancer based on hormonereceptor status (e.g., not expressing the estrogen receptor and/orprogesterone receptor) in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),b) an effective amount of an antimetabolite (such as a nucleosideanalog, for example, gemcitabine), and c) an effective amount of ananthracycline antibiotic (such as epirubicin). In some embodiments, themethod is for treatment of breast cancer in a neoadjuvant setting. Forexample, in some embodiments, there is provided a method of treatinglocally advanced/inflammatory cancer in an individual comprisingadministering to the individual 220 mg/m² paclitaxel/albuminnanoparticle composition (such as Abraxane®) every two weeks; 2000 mg/m²gemcitabine, every two weeks; and 50 mg/m² epirubicin, every two weeks.In some embodiments, there is provided a method of treating breastcancer in an individual in an adjuvant setting, comprising administeringto the individual 175 mg/paclitaxel/albumin paclitaxel/albuminnanoparticle composition (such as Abraxane®) every two weeks, 2000 mg/m²gemcitabine, every two weeks, and 50 mg/m² epirubicin, every two weeks.

In some embodiments, there is provided a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), b) an effective amount of a platinum-basedagent (such as carboplatin), and c) a therapeutic antibody (such asant-HER2 antibody (such as Herceptin®) and anti-VEGF antibody (such asAvastin®)). In some embodiments, there is provided a method of treatingbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®), b) an effective amount of a platinum-basedagent (such as carboplatin), and c) a therapeutic antibody (such asant-HER2 antibody (such as Herceptin®) and anti-VEGF antibody (such asAvastin®)). In some embodiments, the method is for treatment of any ofadvanced breast cancer, metastatic breast cancer, and breast cancer inan adjuvant setting. In some embodiments, there is provided a method oftreating metastatic cancer in an individual, comprising administering tothe individual 75 mg/m² paclitaxel/albumin nanoparticle composition(such as Abraxane®) and carboplatin, AUC=2, wherein the administrationis carried out weekly for three weeks with the fourth week off. In someembodiments, the method further comprises weekly administering about 2-4mg/kg of Herceptin®.

In some embodiments, there is provided a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), b) an effective amount of a platinum-basedagent (such as carboplatin), and c) vinca alkaloid (such as Navelbine®).In some embodiments, there is provided a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®), b) an effective amount of a platinum-basedagent (such as carboplatin), and c) a vinca alkaloid (such asNavelbine®).

In some embodiments, the invention provides a method of treating breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), b) an effective amount of an alkylating agent(such as cyclophosphamide) and c) an anthracycline antibiotic (such asadriamycin). In some embodiments, the invention provides a method oftreating a proliferative disease (such as cancer) in an individual,comprising administering to the individual a) an effective amount of acomposition comprising nanoparticles comprising paclitaxel and analbumin, b) an effective amount of an alkylating agent (such ascyclophosphamide) and c) an anthracycline antibiotic (such asadriamycin). In some embodiments, the method is for treatment of anearly stage breast cancer. In some embodiments, the method is fortreatment of a breast cancer in an adjuvant or a neoadjuvant setting.For example, in some embodiments, there is provided a method of treatingan early stage breast cancer in an individual, comprising administering260 mg/m² paclitaxel/albumin nanoparticle composition (such asAbraxane®), 60 mg/m² adriamycin, and 600 mg/m² cyclophosphamide, whereinthe administration is carried out once every two weeks.

Other embodiments are provided in Table 1. For example, in someembodiments, there is provided a method of treating advanced breastcancer in an individual, comprising administering to the individual a)an effective amount of a composition comprising nanoparticles comprisinga paclitaxel and an albumin (such as Abraxane®), b) an effective amountof carboplatin. In some embodiments, the method further comprisesadministering an effective amount of Herceptin® to the individual. Insome embodiments, there is provided a method of treating metastaticbreast cancer in an individual, comprising administering to theindividual a) an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),b) an effective amount of gemcitabine. In some embodiments, there isprovided a method of treating advanced non-small cell lung cancer in anindividual, comprising administering to the individual a) an effectiveamount of a composition comprising nanoparticles comprising paclitaxeland an albumin (such as Abraxane®), b) an effective amount ofcarboplatin.

In some embodiments, there is provided a composition comprisingnanoparticles comprising a taxane (such as paclitaxel, docetaxel, orortataxel) and a carrier protein (such as albumin) and at least oneother chemotherapeutic agent. The compositions described herein maycomprise effective amounts of the taxane and the chemotherapeutic agentfor the treatment of breast cancer based on hormone receptor status(e.g., not expressing the estrogen receptor and/or progesteronereceptor). In some embodiments, the chemotherapeutic agent and thetaxane are present in the composition at a predetermined ratio, such asthe weight ratios described herein. In some embodiments, the inventionprovides a synergistic composition of an effective amount of acomposition comprising nanoparticles comprising a taxane (such aspaclitaxel, docetaxel, or ortataxel) and an effective amount of at leastone other chemotherapeutic agent. In some embodiments, the otherchemotherapeutic agent is an anti-VEGF antibody (such as bevacizumab,for example, Avastin®).

In some embodiments, the invention provides pharmaceutical compositionscomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin) for use in the treatment of breast cancer based on hormonereceptor status (e.g., not expressing the estrogen receptor and/orprogesterone receptor), wherein said use comprises simultaneous and/orsequential administration of at least one other chemotherapeutic agent.In some embodiments, the invention provides a pharmaceutical compositioncomprising a chemotherapeutic agent for use in the treatment of breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor), wherein said usecomprises simultaneous and/or sequential administration of a compositioncomprising nanoparticles comprising a taxane and a carrier protein (suchas albumin). In some embodiments, the invention providestaxane-containing nanoparticle compositions and compositions comprisingone other chemotherapeutic agent for simultaneous, and/or sequential usefor treatment of breast cancer based on hormone receptor status (e.g.,not expressing the estrogen receptor and/or progesterone receptor).

Modes of Administration

The composition comprising nanoparticles comprising taxane (alsoreferred to as “nanoparticle composition”) and the chemotherapeuticagent can be administered simultaneously (i.e., simultaneousadministration) and/or sequentially (i.e., sequential administration) inthe methods described above for treating breast cancer based on hormonereceptor status (e.g., not expressing the estrogen receptor and/orprogesterone receptor).

In some embodiments, the nanoparticle composition and thechemotherapeutic agent (including the specific chemotherapeutic agentsdescribed herein) are administered simultaneously. The term“simultaneous administration,” as used herein, means that thenanoparticle composition and the chemotherapeutic agent are administeredwith a time separation of no more than about 15 minute(s), such as nomore than about any of 10, 5, or 1 minutes. When the drugs areadministered simultaneously, the drug in the nanoparticles and thechemotherapeutic agent may be contained in the same composition (e.g., acomposition comprising both the nanoparticles and the chemotherapeuticagent) or in separate compositions (e.g., the nanoparticles arecontained in one composition and the chemotherapeutic agent is containedin another composition). For example, the taxane and thechemotherapeutic agent may be present in a single composition containingat least two different nanoparticles, wherein some of the nanoparticlesin the composition comprise the taxane and a carrier protein, and someof the other nanoparticles in the composition comprise thechemotherapeutic agent and a carrier protein. The invention contemplatesand encompasses such compositions. In some embodiments, only the taxaneis contained in nanoparticles. In some embodiments, simultaneousadministration of the drug in the nanoparticle composition and thechemotherapeutic agent can be combined with supplemental doses of thetaxane and/or the chemotherapeutic agent.

In some embodiments, the nanoparticle composition and thechemotherapeutic agent are administered sequentially. The term“sequential administration” as used herein means that the drug in thenanoparticle composition and the chemotherapeutic agent are administeredwith a time separation of more than about 15 minutes, such as more thanabout any of 20, 30, 40, 50, 60 or more minutes. Either the nanoparticlecomposition or the chemotherapeutic agent may be administered first. Thenanoparticle composition and the chemotherapeutic agent are contained inseparate compositions, which may be contained in the same or differentpackages.

In some embodiments, the administration of the nanoparticle compositionand the chemotherapeutic agent are concurrent, i.e., the administrationperiod of the nanoparticle composition and that of the chemotherapeuticagent overlap with each other. In some embodiments, the administrationof the nanoparticle composition and the chemotherapeutic agent arenon-concurrent. For example, in some embodiments, the administration ofthe nanoparticle composition is terminated before the chemotherapeuticagent is administered. In some embodiments, the administration of thechemotherapeutic agent is terminated before the nanoparticle compositionis administered. The time period between these two non-concurrentadministrations can range from about two to eight weeks, such as aboutfour weeks.

The dosing frequency of the drug-containing nanoparticle composition andthe chemotherapeutic agent may be adjusted over the course of thetreatment, based on the judgment of the administering physician. Whenadministered separately, the drug-containing nanoparticle compositionand the chemotherapeutic agent can be administered at different dosingfrequency or intervals. For example, the drug-containing nanoparticlecomposition can be administered weekly, while a chemotherapeutic agentcan be administered more or less frequently. In some embodiments,sustained continuous release formulation of the drug-containingnanoparticle and/or chemotherapeutic agent may be used. Variousformulations and devices for achieving sustained release are known inthe art.

The nanoparticle composition and the chemotherapeutic agent can beadministered using the same route of administration or different routesof administration. In some embodiments (for both simultaneous andsequential administrations), the taxane in the nanoparticle compositionand the chemotherapeutic agent are administered at a predeterminedratio. For example, in some embodiments, the ratio by weight of thetaxane in the nanoparticle composition and the chemotherapeutic agent isabout 1 to 1. In some embodiments, the weight ratio may be between about0.001 to about 1 and about 1000 to about 1, or between about 0.01 toabout 1 and 100 to about 1. In some embodiments, the ratio by weight ofthe taxane in the nanoparticle composition and the chemotherapeuticagent is less than about any of 100:1, 50:1, 30:1, 10:1, 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1, and 1:1 In some embodiments, the ratio byweight of the taxane in the nanoparticle composition and thechemotherapeutic agent is more than about any of 1:1, 2:1, 3:1, 4:1,5:1, 6:1, 7:1, 8:1, 9:1, 30:1, 50:1, 100:1. Other ratios arecontemplated.

The doses required for the taxane and/or the chemotherapeutic agent may(but not necessarily) be lower than what is normally required when eachagent is administered alone. Thus, in some embodiments, a subtherapeuticamount of the drug in the nanoparticle composition and/or thechemotherapeutic agent are administered. “Subtherapeutic amount” or“subtherapeutic level” refer to an amount that is less than thetherapeutic amount, that is, less than the amount normally used when thedrug in the nanoparticle composition and/or the chemotherapeutic agentare administered alone. The reduction may be reflected in terms of theamount administered at a given administration and/or the amountadministered over a given period of time (reduced frequency).

In some embodiments, enough chemotherapeutic agent is administered so asto allow reduction of the normal dose of the drug in the nanoparticlecomposition required to effect the same degree of treatment by at leastabout any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, or more. Insome embodiments, enough drug in the nanoparticle composition isadministered so as to allow reduction of the normal dose of thechemotherapeutic agent required to affect the same degree of treatmentby at least about any of 5%, 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90%, ormore.

In some embodiments, the dose of both the taxane in the nanoparticlecomposition and the chemotherapeutic agent are reduced as compared tothe corresponding normal dose of each when administered alone. In someembodiments, both the taxane in the nanoparticle composition and thechemotherapeutic agent are administered at a subtherapeutic, i.e.,reduced, level. In some embodiments, the dose of the nanoparticlecomposition and/or the chemotherapeutic agent is substantially less thanthe established maximum toxic dose (MTD). For example, the dose of thenanoparticle composition andlor the chemotherapeutic agent is less thanabout 50%, 40%, 30%, 20%, or 10% of the MTD.

A combination of the administration configurations described herein canbe used. The combination therapy methods described herein may beperformed alone or in conjunction with another therapy, such as surgery,radiation, chemotherapy, immunotherapy, gene therapy, and the like.Additionally, a person having a greater risk of developing theproliferative disease may receive treatments to inhibit or and/or delaythe development of the disease.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be approximately thosealready employed in clinical therapies wherein the chemotherapeuticagent are administered alone or in combination with otherchemotherapeutic agents. Variation in dosage will likely occur dependingon the condition being treated. As described above, in some embodiments,the chemotherapeutic agents may be administered at a reduced level.

The nanoparticle compositions described herein can be administered to anindividual (such as human) via various routes, such as parenterally,including intravenous, intra-arterial, intraperitoneal, intrapulmonary,oral, inhalation, intravesicular, intramuscular, intra-tracheal,subcutaneous, intraocular, intrathecal, or transdermal. For example, thenanoparticle composition can be administered by inhalation to treatconditions of the respiratory tract. The composition can be used totreat respiratory conditions such as pulmonary fibrosis, broncheolitisobliterans, lung cancer, bronchoalveolar carcinoma, and the like. Insome embodiments, the nanoparticle composition is administratedintravenously. In some embodiments, the nanoparticle composition isadministered orally.

The dosing frequency of the administration of the nanoparticlecomposition depends on the nature of the combination therapy and theparticular disease being treated. An exemplary dosing frequency include,but is not limited to, weekly without break; weekly, three out of fourweeks; once every three weeks; once every two weeks; weekly, two out ofthree weeks. See also Table 1.

The dose of the taxane in the nanoparticle composition will vary withthe nature of the combination therapy and the particular disease beingtreated. The dose should be sufficient to effect a desirable response,such as a therapeutic or prophylactic response against a particulardisease. An exemplary dose of the taxane (in some embodimentspaclitaxel) in the nanoparticle composition include, but is not limitedto, about any of 50 mg/m², 60 mg/m². 75 mg/m², 80 mg/m², 90 mg/m², 100mg/m²; 120 mg/m²; 160 mg/m², 175 mg/m², 200 mg/m², 210 mg/m², 220 mg/m²,260 mg/m², and 300 mg/m². For example, the dosage of paclitaxel in ananoparticle composition can be in the range of 100-400 mg/m² when givenon a 3 week schedule, or 50-250 mg/m² when given on a weekly schedule.See also Table 1.

Other exemplary dosing schedules for the administration of thenanoparticle composition (such as paclitaxel/albumin nanoparticlecomposition, for example Abraxane®) include, but are not limited to, 100mg/m², weekly, without break; 75 mg/m² weekly, 3 out of four weeks; 100mg/m², weekly, 3 out of 4 weeks; 125 mg/m², weekly, 3 out of 4 weeks;125 mg/m², weekly, 2 out of 3 weeks; 130 mg/m², weekly, without break;175 mg/m², once every 2 weeks; 260 mg/m², once every 2 weeks; 260 mg/m²,once every 3 weeks; 180-300 mg/m², every three weeks; 60-175 mg/m²,weekly, without break. In addition, the taxarie (alone or in combinationtherapy) can be administered by following a metronomic dosing regimedescribed herein.

Exemplary dosing regimes for the combination therapy of nanoparticlecomposition (such as paclitaxel/albumin nanoparticle composition, forexample Abraxane®) and other agents include, but are not limited to, 125mg/m² weekly, two out of three weeks, plus 825 mg/m² Xeloda®, daily. Thedose of the nanoparticle composition (such as paclitaxel/albuminnanoparticle composition, for example Abraxane®) and Xeloda® may varydepending on the particular disease or the patient being treated. Thedose should be sufficient to effect a desirable response, such as atherapeutic or prophylactic response against a particular disease. Anexemplary dose of the nanoparticle composition (in some embodimentspaclitaxel/albumin nanoparticle composition, for example Abraxane®) inthe combination therapy includes, but is not limited to, about any of100 mg/m², 125 mg/m², 200 mg/m², and 260 mg/m². For example, the dosageof paclitaxel in a nanoparticle composition can be in the range of50-300 mg/m² when given on a weekly schedule, with or without breaks. Anexemplary dose of Xeloda® in the combination therapy includes, but isnot limited to, about any of 550 mg/m², 650 mg/m², 825 mg/m², 850 mg/m²,1000 mg/m² and 1250 mg/m². For example, the dosage of Xeloda® can be inthe range of 500-2500 mg/m² when given on a daily schedule, with orwithout breaks.

Exemplary dosing regimes for the combination therapy of nanoparticlecomposition (such as paclitaxel/albumin nanoparticle composition, forexample Abraxane®) and other agents include, but are not limited to, 260mg/m² once every two weeks plus 60 mg/m² adriamycin and 600 mg/m²cyclophosphamide, once every two weeks; 220-340 mg/m² once every threeweeks, plus carboplatin, AUC=6, once every three weeks; 100-150 mg/m²weekly, plus carboplatin, AUC=6, once every three weeks; 175 mg/m2 onceevery two weeks, plus 2000 mg/m² gemcitabine and 50 mg/m² epirubicin,once every two weeks; and 75 mg/m² weekly, three out of four weeks, pluscarboplatin, AUC=2, weekly, three out of four weeks.

In some embodiments, the nanoparticle composition of the taxane and thechemotherapeutic agent is administered according to any of the dosingregimes described in Table 1.

In some embodiments, there is provided a method of treating breastcancer in an individual comprising administering to the individual: a)an effective amount of a composition comprising nanoparticles comprisinga taxane (such as paclitaxel) and an albumin, and b) an effective amountof at least one other chemotherapeutic agent as provided in Rows 1 to 35in Table 1. In some embodiments, the administration of the nanoparticlecomposition and the chemotherapeutic agent may be any of the dosingregimes as indicated in Rows 1 to 35 in Table 1. In some embodiments,there is provided a method of treating metastatic breast cancer in anindividual comprising administering to the individual: a) an effectiveamount of a composition comprising nanoparticles comprising a taxane(such as paclitaxel) and an albumin, and b) an effective amount of atleast one other chemotherapeutic agent as provided in Rows 2, 4-8, and10-15 in Table 1. In some embodiments, the administration of thenanoparticle composition and the chemotherapeutic agent may be any ofthe dosing regimes as indicated in Rows 2, 4-8, and 10-15 in Table 1.

In some embodiments, there is provided a method of treating advancedbreast cancer in an individual comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Rows 1 and 16 in Table 1. In some embodiments, theadministration of the nanoparticle composition and the chemotherapeuticagent may be any of the dosing regimes as indicated in Rows 1 and 16 inTable 1. In some embodiments, there is provided a method of treatingstage IV breast cancer in an individual comprising administering to theindividual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Row 3 in Table 1. In some embodiments, the administrationof the nanoparticle composition and the chemotherapeutic agent may bethe dosing regime as indicated in Row 3 in Table 1.

In some embodiments, there is provided a method of treating breastcancer in an individual in an adjuvant setting comprising administeringto the individual: a) an effective amount of a composition comprisingnanoparticles comprising a taxane (such as paclitaxel) and an albumin,and b) an effective amount of at least one other chemotherapeutic agentas provided in Rows 18 to 24 in Table 1. In some embodiments, theadministration of the nanoparticle composition and the chemotherapeuticagent may be any of the dosing regimes as indicated in Rows 18 to 24 inTable 1.

In some embodiments, there is provided a method of treating breastcancer in an individual in a neoadjuvant setting comprisingadministering to the individual: a) an effective amount of a compositioncomprising nanoparticles comprising a taxane (such as paclitaxel) and analbumin, and b) an effective amount of at least one otherchemotherapeutic agent as provided in Rows 25 to 35 in Table 1. In someembodiments, the administration of the nanoparticle composition and thechemotherapeutic agent may be any of the dosing regimes as indicated inRows 25 to 35 in Table 1.

In some embodiments, there is provided a method of treating solid tumor(including advanced solid tumor) in an individual comprisingadministering to the individual: a) an effective amount of a compositioncomprising nanoparticles comprising a taxane (such as paclitaxel) and analbumin, and b) an effective amount of at least one otherchemotherapeutic agent as provided in Rows 36 to 39 in Table 1. In someembodiments, the administration of the nanoparticle composition and thechemotherapeutic agent may be any of the dosing regimes as indicated inRows 36 to 39 in Table 1.

TABLE 1 Row Study therapy No. Combination Regime/Dosage type Protocoltitle 1. ABX + ABX: 100 mg/m² D1, 8, 15 Advanced A phase II study ofCarboplatin + q4wk × 6 HER2+ Breast weekly dose-dense Herceptin ® Carbo:AUC = 2 D1, 8, 15 Cancer nanoparticle paclitaxel q4wk × 6 (ABI-007)Herceptin ®: 4 mg/kg on wk 1, 2 carboplatin ™, with mg/kg all subsequentweeks Herceptin ® as first or second-line therapy of advanced HER2+breast cancer 2. ABX alone ABX: 125 mg/m² Metastatic Phase II trial ofweekly (+Herceptin ®) qwk × 3/4 Breast Cancer Abraxane ® monotherapy for1st- line MBC (plus Herceptin ® in HER2+ pts) 3. ABX + L1: ABX: 80 mg/mStage IV Phase I-II study weekly Navelbine ® Nav: 15 mg/m² Breast CancerABX + Navelbine ®, (±G-CSF) L2: ABX: 90 mg/m² with or without G-CSF,Nav: 20 mg/m² in stage IV breast L3: ABX: 100 mg/m² cancer Nav: 22.5mg/m² L4: ABX: 110 mg/m² Nav: 25 mg/m2 L5: ABX: 125 mg/m² Nav: 25 mg/m²qwk all levels 4. ABX + Xeloda ® ABX: 125 mg/m² qwk × 2/3 MetastaticPhase II 1st-line ABX + Xeloda ®: 825 mg/m² D1-14 Breast Cancer Xeloda ®MBC trial q3wk 5. ABX + Metastatic Phase I/II trial ABX AnthracyclineBreast Cancer plus Doxil ® for MBC plus limited PK 6. ABX + ABX: 125mg/m² Metastatic Randomized Phase II Gemcitabine Gem: 1000 mg/m² BreastCancer Trial of Weekly nab qwk × 2/3 (nanoparticle albuminbound)-Paclitaxel (nab-paclitaxel) in Combination with Gemcitabine inPatients with HER2 Negative Metastatic Breast Cancer 7. ABX + LapatinibMetastatic Phase I/II Abraxane ® + Breast Cancer GW572016 8. ABX +Lapatinib ABX: 100 mg/m² qwk × 3/4 Metastatic Phase I dose escalationLapatinib: starting at Breast Cancer study of a 2 day oral 1000 mg/d × 2days lapatinib chemosensitization pulse given prior to weeklyintravenous Abraxane ® in patients with advanced solid tumors 9. ABX +FEC ABX: 220 mg/m2 q2wk × 6 Breast Cancer Phase II preoperative(+Herceptin ®) followed by trial of Abraxane ® FEC: 4 cycles(+Herceptin ® for followed by FEC HER2+ pts) (+Herceptin ® asappropriate) in breast cancer 10. ABX + ABX: 100 mg/m² qwk D1, 8, 15Metastatic Phase II safety and Carboplatin + Carbo: AUC = 2 qwk D1, 8,15 Breast Cancer tolerability study of Avastin ® Avastin ®: 10 mg/m²q2wk (HER2−, ER−, Abraxane ®, Avastin ® PR−) and carboplatin in triple,negative metastatic breast cancer patients 11. ABX + Avastin ® ABX: 130mg/m² qwk + Metastatic Three arm phase II trial Avastin ® Breast Cancerin l^(st) line HER2− vs negative MBC patients ABX: 260 mg/m² q2wk +Avastin ® vs ABX: 260 mg/m² q3wk + Avastin ® 12. ABX + Avastin ® ABX:125 mg/m² qwk × 3/4 + Metastatic Single arm study of Avastin ® BreastCancer Abraxane ® and Avastin ® in 1^(st) line MBS 13. ABX + Avastin ®ABX + Avastin ® qwk Metastatic Randomized Phase III vs Breast Cancertrial in 1^(st) line and 2^(nd) Taxol ® + Avastin ® qwk line MBC withbiological correlates analysis 14. ABX + Xeloda ® + Metastatic Phase IIAbraxane ® in Lapatinib Breast Cancer combination with Xeloda ® andLapatinib for metastatic breast cancer 15. ABX + ABX: 3000 mg/m² D1 q3wkMetastatic Single arm Phase II Gemcitabine Gem: 12.50 mg/m² D1, 8 q3vvkBreast Cancer study of Abraxane ® and gemcitabine for 1^(st) line MBC16. ABX + RAD001 Advanced Phase I/II study of Breast Cancer Abraxane ®in combination with RAD001 in patients with advanced breast cancer 17.ABX + Sutent ® Breast Cancer Phase I study of Abraxane ® in combinationwith Sutent ® 18. ABX + AC + AC + G-CSF q2wk × 4 Breast Cancer-Abraxane ® in dose- G-CSF followed by Adjuvant dense adjuvant(+Herceptin ®) ABX: 260 mg/m² q2wk × 4 chemotherapy for early(+Herceptin ® for HER2+ pts) stage breast cancer 19. ABX + AC + Dosedense AC + G-CSF Breast Cancer- Phase II pilot adjuvant G-CSF followedby ABX Adjuvant trial of Abraxane ® in (+Herceptin ®) (+Herceptin ® forHER2+ pts) breast cancer qwk 20. ABX + AC AC followed by ABX: 260 BreastCancer- Adjuvant Dose dense mg/m² Adjuvant Registrational Trial vs ACfollowed by Taxol ® Rx length 16 wks 21. ABX + AC AC q2wk followed byBreast Cancer- Phase II dose dense (+G-CSF) ABX: 260 mg/m² + G-CSF q2wkAdjuvant pilot adjuvant study of Rx length 16 wks Abraxane ® in breastcancer 22. ABX + AC Dose dense AC followed by Breast Cancer- Pilotadjuvant breast (+Avastin ®) ABX (+Avastin ® in HER2+ pts) Adjuvantcancer study 23. ABX + AC AC Breast Cancer- BIG study: Dose densefollowed by ABX Adjuvant vs standard adjuvant q2wk or q3wk chemotherapy24. ABX (ABI-007) + AC followed by Breast Cancer- Phase II—Pilot StudyAC + Neulasta ® ABX q2wk × 4 Adjuvant Evaluating the Safety of aDose-Dense Regime— AC × 4 => ABI-007 × 4 Q 2 WEEKS + Neulasta ®—Given asAdjuvant Chemotherapy of High- Risk Women with Early Breast Cancer 25.ABX + FEC ABX: 100 mg/m² qwk × 12 Locally A Phase II Study of(+Herceptin ®) followed by Advanced Breast Neoadjuvant 5-FU: 500 mg/m²q3wk Cancer- Chemotherapy with Epirubicin: 100 mg/m² NeoadjuvantSequential Weekly (without Herceptin ®) Nanoparticle Albumin or BoundPaclitaxel Epirubicin: 75 mg/m² (Abraxane ®) Followed (with Herceptin ®for HER2+ by 5-Fluorouracil, pts) Epirubicin, Cyclophosphamide: 500mg/m² Cyclophosphamide q3wk (FEC) in Locally Advanced Breast Cancer 26.ABX + Arm 1: Neoadjuvant: Gem: 2000 Breast Cancer- Phase II Trial ofDose Gemcitabine + mg/m², ABX: 175 mg/m², Epi Neoadjuvant DenseNeoadjuvant Epirubicin 50 mg/m² Gemcitabine, q2wk × 6 Epirubicin,ABI-007 Arm 2: Adjuvant: Gem: 2000 (GEA) in Locally mg/m², ABX: 220mg/m² Advanced or q2wk × 4 Inflammatory Breast Cance 27. ABX + ABX: 260mg/m² q2wk + Breast Cancer- Phase II Multi-center Herceptin ®Herceptin ® Neoadjuvant study neoadjuvant. followed by Navelbine ® +Herceptin ® 28. ABX + TAC Breast Cancer- 3 arms Randomized Carboplatinvs Neoadjuvant dose dense phase II (+Herceptin ®) + AC followed by ABX +carbo trial of neoadjuvant AC vs chemotherapy in AC followed by ABX +carbo + patients with breast Herceptin ® cancer 29. ABX + ABX: 260 mg/m²q3wk × 4 Breast Cancer- Phase II neoadjuvant Capecitabine Xeloda ® 850mg/m² D1-14 Neoadjuvant trial of Abraxane ® and q3wk × 4 capecitabine inlocally advanced breast cancer 30. ABX + ABX qwk Breast Cancer- PhaseI/II trial of Carboplatin carbo qwk + Neoadjuvant neoadjuvant(+Avastin ®) Avastin ® in HER2+ pts chemotherapy (NCT) with weeklynanoparticle paclitaxel (ABI-007, Abraxane ®) in combination withcarboplatin and Avastin ® in clinical stage I-III. 31. ABX + ABX: 100mg/m² qwk × 3/4 Breast Cancer- Phase II study of Carboplatin + Carbo:AUC = 5 + Neoadjuvant weekly bevacizumab Herceptin ® + Herceptin ® +administered with Avastin ® Avastin ® weekly trastuzumab, 4 week cycle ×6 ABI-007, and carboplatin as preoperative therapy in HER2-neu geneamplified breast cancer tumors 32. ABX + Lapatinib ABX: 260 mg/m² q3wkBreast Cancer Pilot neoadjuvant trial Lapatinib: 1000 mg/day Neoadjuvantwith combination of ABI-007 (Abraxane ®) and GW572016 (Lapatinib) 33.ABX + ABX: 200 mg/m² Breast Cancer- Phase II neoadjuvant Capecitabineq3wk × 4 Neoadjuvant trial of Abraxane ® and Xeloda ®: 1000 mg/m²capecitabine in locally D1-14 q3wk × 4 advanced breast cancer 34. ABX ±ABX qwk ± Avastin ® followed Breast Cancer- Phase III trial ofAvastin ® + AC by A qwk + C daily Neoadjuvant paclitaxel vs Abraxane ®(+G-CSF) vs with or without Taxol ® qwk ± Avastin ® Avastin ® infollowed by A qwk + C daily combination with doxorubicin andcyclophosphamide plus G-CSF 35. ABX + AC ABX followed by AC BreastCancer- Phase II neoadjuvant Neoadjuvant trial with gene expressionanalyses 36. ABX + ABX: 100 mg/m² qwk Solid Tumors Phase I Study ofRapamycin Rapamycin: 5-40 mg dose Rapamycin in escalation Combinationwith Abraxatin ® in Advanced Solid Tumors 37. ABX + Satraplatin SolidTumors Phase I trial of Abraxane ® and Satraplatin 38. ABX + ABX: 180,220, 260, 300, 340 Advanced Solid Phase I Trial of Gemcitabine mg/m²q3wk Tumors Abraxane ® in Gemcitabine: 1000 mg/m² D1 combination and D8with Gemcitabine 39. ABX + Gefitinib ABX: 100 mg/m² qwk × 3/4 AdvancedSolid Phase I dose escalation Gefitinib starting at 1000 mg/d × 2 Tumorsstudy of gefitinib chemosensitization pulse given prior to weeklyAbraxane ®

As used in herein (for example in Table 1), ABX refers to Abraxane®;GW572016 refers to lapatinib; Xel refers to capecitabine or Xeloda®;bevacizumab is also known as Avastin®; trastuzurnab is also known asHerceptin®; pemtrexed is also known as Alimta®; cetuximab is also knownas Erbitux®, gefitinib is also known as Iressa®; FEC refers to acombination of 5-fluorouracil, Epirubicin and Cyclophosphamide; ACrefers to a combination of Adriamycin plus Cyclophosphamide; TAC refersto a FDA approved adjuvant breast cancer regime; RAD001 refers to aderivative of rapamycin.

As used herein (for example in Table 1), AUC refers to area under curve;q4wk refers to a dose every 4 weeks; q3wk refers to a dose every 3weeks; q2wk refers to a dose every 2 weeks; qwk refers to a weekly dose;qwk×¾ refers to a weekly dose for 3 weeks with the 4^(th) week off;qwk×⅔ refers to a weekly dose for 2 weeks with the 3^(rd) week off

Combination Therapy with Radiation Therapy and Surgery

In another aspect, the present invention provides a method of treatingbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) comprising a firsttherapy comprising administering a taxane (particularly nanoparticlescomprising a taxane) and a carrier protein and a second therapycomprising radiation and/or surgery.

In some embodiments, the hormone receptor status is low for one or morehormone receptors such as the estrogen receptor or the progesteronereceptor. In some embodiments, the individual is likely more responsiveto the therapy if hormone receptor status is low for both estrogenreceptor and progesterone receptor. In some embodiments, the hormonereceptor status does not express (i.e., is negative for) one or morehormone receptors such as the estrogen receptor (ER) or the progesteronereceptor (PgR). In some embodiments, the hormone receptor status of thebreast cancer tissue does not express (i.e., is negative for) both theestrogen receptor (ER) and the progesterone receptor (PgR). In someembodiments, the individual is likely more responsive to the therapy ifhormone receptor status is negative for both estrogen receptor andprogesterone receptor. In some embodiments, the individual expresses(i.e., is positive for) either the estrogen receptor or the progesteronereceptor. In some embodiments, the individual expresses (i.e., ispositive for) both the estrogen receptor and the progesterone receptor.In some embodiments, the individual is likely less responsive to therapyif the hormone receptor status is positive for the estrogen receptorand/or the progesterone receptor.

In some embodiments, the breast cancer tissue further expresses HER2(HER2+) some embodiments, the breast cancer tissue further does notexpress HER2 (HER2−).

In some embodiments, the method comprises: a) a first therapy comprisingadministering to the individual a composition comprising nanoparticlescomprising an effective amount of a taxane and a carrier protein (suchas albumin) and b) a second therapy comprising radiation therapy,surgery, or combinations thereof. In some embodiments, the taxane iscoated with the carrier protein (such as albumin). In some embodiments,the second therapy is radiation therapy. In some embodiments, the secondtherapy is surgery.

In some embodiments, the method comprises a) a first therapy comprisingadministering to the individual a composition comprising nanoparticlescomprising paclitaxel and an albumin; and b) a second therapy comprisingradiation therapy, surgery, or combinations thereof. In someembodiments, the second therapy is radiation therapy. In someembodiments, the second therapy is surgery. In some embodiments, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm. In some embodiments, the paclitaxel/albuminnanoparticle composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some embodiments, the weight ratio ofthe albumin to paclitaxel in the composition is about 18:1 or less, suchas about 9:1 or less. In some embodiments, the paclitaxel is coated withalbumin. In some embodiments, the paclitaxel/albumin nanoparticles havean average diameter of no greater than about 200 nm and thepaclitaxel/albumin composition is substantially free (such as free) ofsurfactant (such as Cremophor). In some embodiments, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel is coated with albumin. In someembodiments, the nanoparticle composition is Abraxane®.

The administration of the nanoparticle composition may be prior to theradiation and/or surgery, after the radiation and/or surgery, orconcurrent with the radiation and/or surgery. For example, theadministration of the nanoparticle composition may precede or follow theradiation and/or surgery therapy by intervals ranging from minutes toweeks. In some embodiments, the time period between the first and thesecond therapy is such that the taxane and the radiation/surgery wouldstill be able to exert an advantageously combined effect on the cell.For example, the taxane (such as paclitaxel) in the nanoparticlecomposition may be administered less than about any of 1, 3, 6, 9, 12,18, 24, 48, 60, 72, 84, 96, 108, 120 hours prior to the radiation and/orsurgery. In some embodiments, the nanoparticle composition isadministered less than about 9 hours prior to the radiation and/surgery.In some embodiments, the nanoparticle composition is administered lessthan about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days prior to theradiation/surgery. In some embodiments, the taxane (such as paclitaxel)in the nanoparticle composition is administered less than about any of1, 3, 6, 9, 12, 18, 24, 48, 60, 72, 84, 96, 108, or 120 hours after theradiation and/or surgery. In some embodiments, it may be desirable toextend the time period for treatment significantly, where several daysto several weeks lapse between the two therapies.

Radiation contemplated herein includes, for example, γ-rays, X-rays(external beam), and the directed delivery of radioisotopes to tumorcells. Other forms of DNA damaging factors are also contemplated such asmicrowaves and UV irradiation are also contemplated. Radiation may begiven in a single dose or in a series of small doses in adose-fractionated schedule. The amount of radiation contemplated hereinranges from about 1 to about 100 Gy, including, for example, about 5 toabout 80, about 10 to about 50 Gy, or about 10 Gy. The total dose may beapplied in a fractioned regime. For example, the regime may comprisefractionated individual doses of 2 Gy. Dosage ranges for radioisotopesvary widely, and depends on the half-life of the isotope and thestrength and type of radiation emitted.

When the radiation comprises use of radioactive isotopes, the isotopemay be conjugated to a targeting agent, such as a therapeutic antibody,which carries the radionucleotide to the target tissue. Suitableradioactive isotopes include, but are not limited to, astatine²¹¹,carbon¹⁴, chromium⁵¹, chlorine³⁶, iron⁵⁷, cobalt⁵⁸, copper⁶⁷, Eu¹⁵²,gallium⁶⁷, hydrogen³, iodine¹²³, iodine¹³¹, indium¹¹¹, iron⁵⁹,phosphorus³², rhenium¹⁸⁶, selenium⁷⁵, sulphur³⁵, technicium^(99m),and/or yttrium⁹⁰.

In some embodiments, enough radiation is applied to the individual so asto allow reduction of the normal dose of the taxane (such as paclitaxel)in the nanoparticle composition required to effect the same degree oftreatment by at least about any of 5%, 10%, 20%, 30%, 50%, 60%, 70%,80%, 90%, or more. In some embodiments, enough taxane in thenanoparticle composition is administered so as to allow reduction of thenormal dose of the radiation required to effect the same degree oftreatment by at least about any of 5%, 10%, 20%, 30%, 50%, 60%, 70%,80%, 90%, or more. In some embodiments, the dose of both the taxane(such as paclitaxel) in the nanoparticle composition and the radiationare reduced as compared to the corresponding normal dose of each whenused alone.

In some embodiments, the combination of administration of thenanoparticle composition and the radiation therapy producesupra-additive effect. In some embodiments, the taxane (such aspaclitaxel) in the nanoparticle composition is administered once at thedose of 90 mg/kg, and the radiation is applied five times at 80 Gydaily.

Surgery described herein includes resection in which all or part ofcancerous tissue is physically removed, exercised, and/or destroyed.Tumor resection refers to physical removal of at least part of a tumor.In addition to tumor resection, treatment by surgery includes lasersurgery, cryosurgery, electrosurgery, and micropically controlledsurgery (Mohs surgery). Removal of superficial surgery, precancers, ornormal tissues are also contemplated.

The radiation therapy and/or surgery may be carried out in addition tothe administration of chemotherapeutic agents. For example, theindividual may first be administered with a taxane-containingnanoparticle composition and at least one other chemotherapeutic agent,and subsequently be subject to radiation therapy and/or surgery.Alternatively, the individual may first be treated with radiationtherapy and/or surgery, which is then followed by the administration ofa nanoparticle composition and at least one other chemotherapeuticagent. Other combinations are also contemplated.

Administration of nanoparticle compositions disclosed above inconjunction with administration of chemotherapeutic agent is equallyapplicable to those in conjunction with radiation therapy and/orsurgery.

In some embodiments, the invention provides pharmaceutical compositionscomprising nanoparticles comprising a taxane (such as paclitaxel) and acarrier protein (such as albumin) for use in the treatment of breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor), wherein said usecomprises a second therapy comprising radiation therapy, surgery, orcombinations thereof.

Metronomic Therapy

The invention also provides metronomic therapy regime for treatment ofbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor). There is provided amethod of administering to an individual a composition comprisingnanoparticles comprising a taxane (such as paclitaxel, docetaxel, orortataxel) and a carrier protein (such as albumin) based on a metronomicdosing regime. The methods are applicable to methods of treatment,delaying development, and other clinical settings and configurationsdescribed herein.

In some embodiments, the hormone receptor status is low for one or morehormone receptors such as the estrogen receptor or the progesteronereceptor. In some embodiments, the individual is likely more responsiveto the therapy if hormone receptor status is low for both estrogenreceptor and progesterone receptor. In some embodiments, the hormonereceptor status does not express (i.e., is negative for) one or morehormone receptors such as the estrogen receptor (ER) or the progesteronereceptor (PgR). In some embodiments, the hormone receptor status of thebreast cancer tissue does not express (i.e., is negative for) both theestrogen receptor (ER) and the progesterone receptor (PgR). In someembodiments, the individual is likely more responsive to the therapy ifhormone receptor status is negative for both estrogen receptor andprogesterone receptor. In some embodiments, the individual expresses(i.e., is positive for) either the estrogen receptor or the progesteronereceptor. In some embodiments, the individual expresses (i.e., ispositive for) both the estrogen receptor and the progesterone receptor.In some embodiments, the individual is likely less responsive to therapyif the hormone receptor status is positive for the estrogen receptorand/or the progesterone receptor.

In some embodiments, the breast cancer tissue further expresses HER2(HER2+). In some embodiments, the breast cancer tissue further does notexpress HER2 (HER2−).

Metronomic dosing regime” used herein refers to frequent administrationof a taxane at without prolonged breaks at a dose below the establishedmaximum tolerated dose via a traditional schedule with breaks(hereinafter also referred to as a “standard MTD schedule” or a“standard MTD regime”). In metronomic dosing, the same, lower, or highercumulative dose over a certain time period as would be administered viaa standard MTD schedule may ultimately be administered. In some cases,this is achieved by extending the time frame and/or frequency duringwhich the dosing regime is conducted while decreasing the amountadministered at each dose. Generally, the taxane administered via themetronomic dosing regime of the present invention is better tolerated bythe individual. Metronomic dosing can also be referred to as maintenancedosing or chronic dosing.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25% to about25% of its maximum tolerated dose following a traditional dosing regime.In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin, wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25% to about 25% of its maximumtolerated dose following a traditional dosing regime.

In some embodiments, the dosing of the taxane (such as paclitaxel) inthe nanoparticle composition per administration is less than about anyof 1%, 2%, 3&, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,18%, 20%, 22%, 24%, or 25% of the MTD for the same taxane (such aspaclitaxel) in the same formulation following a given traditional dosingschedule. Traditional dosing schedule refers to the dosing schedule thatis generally established in a clinical setting. For example, thetradition dosing schedule for Abraxane® is a three-weekly schedule,i.e., administering the composition every three weeks.

In some embodiments, the dosing of the taxane (such as paclitaxel) peradministration is between about 0.25% to about 25% of the correspondingMTD value, including for example any of about 0.25% to about 20%, about0.25% to about 15%, about 0.25% to about 10%, about 0.25% to about 20%,and about 0.25% to about 25%, of the corresponding MTD value. The MTDvalue for a taxane following a traditional dosing schedule is known orcan be easily determined by a person skilled in the art. For example,the MTD value when Abraxane® is administered following a traditionalthree-week dosing schedule is about 300 mg/m².

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25 mg/m² toabout 25 mg/m². In some embodiments, there is provided a method ofadministering a composition comprising nanoparticles comprisingpaclitaxel and an albumin, wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25 mg/m² toabout 25 mg/m².

In some embodiments, the dose of the taxane (such as paclitaxel) at eachadministration is less than about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 18, 20, 22, 25, and 30 mg/m². For example, the dose ofthe taxane (such as paclitaxel) can range from about 0.25 mg/m² to about30 mg/m², about 0.25 mg/m² to about 25 mg/m², about 0.25 mg/m² to about15 mg/m², about 0.25 mg/m² to about 10 mg/m², and about 0.25 mg/m² toabout 5 mg/m².

Dosing frequency for the taxane (such as paclitaxel) in the nanoparticlecomposition includes, but is not limited to, at least about any of oncea week, twice a week, three times a week, four times a week, five timesa week, six times a week, or daily. Typically, the interval between eachadministration is less than about a week, such as less than about any of6, 5, 4, 3, 2, or 1 day. In some embodiments, the interval between eachadministration is constant. For example, the administration can becarried out daily, every two days, every three days, every four days,every five days, or weekly. In some embodiments, the administration canbe carried out twice daily, three times daily, or more frequent.

The metronomic dosing regimes described herein can be extended over anextended period of time, such as from about a month up to about threeyears. For example, the dosing regime can be extended over a period ofany of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, and 36months. Generally, there are no breaks in the dosing schedule.

The cumulative dose of the taxane (such as paclitaxel) administered bythe metronomic regime may be higher than that of the taxane administeredaccording to a standard MTD dosing schedule over the same time period.In some embodiments, the cumulative dose of the taxane administered bythe metronomic regime equals to or is lower than that of the taxaneadministered according to a standard MTD dosing schedule over the sametime period.

It is understood that the teaching provided herein is for examples only,and that metronomic dosing regime can be routinely designed inaccordance with the teachings provided herein and based upon theindividual standard MTD schedule, and that the metronomic dosing regimeused in these experiments merely serves as one example of possiblechanges in dosing interval and duration which are made to a standard MTDschedule to arrive at an optimal metronomic dosing regime.

The metronomic dosing regime described herein may be used alone as atreatment of a proliferative disease, or carried out in a combinationtherapy context, such as the combination therapies described herein. Insome embodiments, the metronomic therapy dosing regime may be used incombination or conjunction with other established therapies administeredvia standard MTD regimes. By “combination or in conjunction with” it ismeant that the metronomic dosing regime of the present invention isconducted either at the same time as the standard MTD regime ofestablished therapies, or between courses of induction therapy tosustain the benefit accrued to the individual by the induction therapy,the intent is to continue to inhibit tumor growth while not undulycompromising the individual's health or the individual's ability towithstand the next course of induction therapy. For example, ametronomic dosing regime may be adopted after an initial short course ofMTD chemotherapy.

The nanoparticle compositions administered based on the metronomicdosing regime described herein can be administered to an individual(such as human) via various routes, such as parenterally, includingintravenous, intra-arterial, intrapulmonary, oral, inhalation,intravesicular, intramuscular, intra-tracheal, subcutaneous,intraocular, intrathecal, or transdermal. For example, the nanoparticlecomposition can be administered by inhalation to treat conditions of therespiratory tract. The composition can be used to treat respiratoryconditions such as pulmonary fibrosis, broncheolitis obliterans, lungcancer, bronchoalveolar carcinoma, and the like. In some embodiments,the nanoparticle composition is administered orally.

Some exemplary embodiments are provided below.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25% to about25% of its maximum tolerated dose following a traditional dosing regime.In some embodiments, the taxane is coated with the carrier protein (suchas albumin). In some embodiments, the dose of the taxane peradministration is less than about any of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, 10%, 11%, 12%, 13%, 14%, 15%, 18%, 20%, 22%, 24%, or 25% of themaximum tolerated dose. In some embodiments, the taxane is administeredat least about any of 1×, 2×, 3×, 4×, 5×, 6×, 7× (i.e., daily) a week.In some embodiments, the intervals between each administration are lessthan about any of 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, and 1day. In some embodiments, the taxane is administered over a period of atleast about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36months.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin, wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25% to about 25% of its maximumtolerated dose following a traditional dosing regime. In someembodiments, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm. In some embodiments, thepaclitaxel/albumin nanoparticle composition is substantially free (suchas free) of surfactant (such as Cremophor), In some embodiments, theweight ratio of the albumin to paclitaxel in the composition is about18:1 or less, such as about 9:1 or less. In some embodiments, thepaclitaxel is coated with albumin. In some embodiments, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel/albumin composition issubstantially free (such as free) of surfactant (such as Cremophor). Insome embodiments, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm and the paclitaxel is coatedwith albumin. In some embodiments, the nanoparticle composition isAbraxane®.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein the nanoparticle composition isadministered over a period of at least one month, wherein the intervalbetween each administration is no more than about a week, and whereinthe dose of the taxane at each administration is about 0.25 mg/m² toabout 25 mg/m². In some embodiments, the taxane is coated with thecarrier protein (such as albumin). In some embodiments, the dose of thetaxane per administration is less than about any of 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 18, 20, 22, and 25 mg/m². In someembodiments, the taxane is administered at least about any of 1×, 2×,3×, 4×, 5×, 6×, 7× (i.e., daily) a week. In some embodiments, theintervals between each administration are less than about any of 7 days,6 days, 5 days, 4 days, 3 days, 2 days, and 1 day. In some embodiments,the taxane is administered over a period of at least about any of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30 and 36 months.

In some embodiments, there is provided a method of administering acomposition comprising nanoparticles comprising paclitaxel and analbumin, wherein the nanoparticle composition is administered over aperiod of at least one month, wherein the interval between eachadministration is no more than about a week, and wherein the dose of thetaxane at each administration is about 0.25 mg/m² to about 25 mg/m². Insome embodiments, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm. In some embodiments, thepaclitaxel/albumin nanoparticle composition is substantially free (suchas free) of surfactant (such as Cremophor). In some embodiments, theweight ratio of the albumin to paclitaxel in the composition is about18:1 or less, such as about 9:1 or less. In some embodiments, thepaclitaxel is coated with albumin. In some embodiments, thepaclitaxel/albumin nanoparticles have an average diameter of no greaterthan about 200 nm and the paclitaxel/albumin composition issubstantially free (such as free) of surfactant (such as Cremophor). Insome embodiments, the paclitaxel/albumin nanoparticles have an averagediameter of no greater than about 200 nm and the paclitaxel is coatedwith albumin. In some embodiments, the nanoparticle composition isAbraxane®.

In some embodiments, the Abraxane® (or other paclitaxel/albuminnanoparticle compositions) is administered at the dose of about 3 mg/kgto about 10 mg/kg daily. In some embodiments, the Abraxane® isadministered at the dose of about 6 mg/kg to about 10 mg/kg daily. Insome embodiments, the Abraxane® is administered at the dose of about 6mg/kg daily. In some embodiments, Abraxane® is administered at the doseof about 3 mg/kg daily.

The invention also provides compositions for use in the metronomicregime(s) described herein. In some embodiments, there is provided acomposition comprising nanoparticles comprising a taxane and a carrierprotein (such as albumin), wherein said composition is administered toan individual via a metronomic dosing regime, such as the dosing regimedescribed herein.

Other Aspects of the Invention

In another aspects, there are provided methods of treating breast cancerbased on hormone receptor status (e.g., not expressing the estrogenreceptor and/or progesterone receptor) comprising administering acomposition comprising nanoparticles comprising a taxane (includingpachiaxel, docetaxel, or ortataxel) and a carrier protein (such asalbumin). In some embodiments, there is provided a method of treatingbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor) comprising administeringa composition comprising nanoparticles comprising ortataxel and acarrier protein (such as albumin).

In some embodiments, there is provided a method of treating cancercomprising administering a composition comprising nanoparticlescomprising paclitaxel, wherein the nanoparticle composition isadministered according to any of the dosing regimes described in Table2. In some embodiments, the cancer is a Taxane refractory metastaticbreast cancer.

TABLE 2 Row Study therapy No. Combination Regimen/Dosage type Protocoltitle 1. ABX alone ABX: 125 mg/m² qwk × 3/4 Metastatic Phase II studywith Breast Cancer weekly Abraxane ® treatment in taxane- refractory MBCpatients 2. ABX alone Arm 1: ABX 130 mg/m² qwk Metastatic 3-arm phase IItrial in 1st- Arm 2: ABX 260 mg/m² q2wk Breast Cancer line Her-2− MBCpatients. Arm 3: ABX 260 mg/m² q3wk 3. ABX alone ABX: 260 mg/m² q3wkMetastatic Phase II Controlled, (Capxol) vs Breast Cancer Randomized,Open Label Taxol: 175 mg/m² q3wk Study to Evaluate the Efficacy andSafety of Capxol (a Cremophor- Free Nanoparticle Paclitaxel) andcremophor-formulated paclitaxel injection in Patient with MetastaticBreast Cancer 4. ABX alone Arm 1: ABX weekly Metastatic 3-arm phase IItrial in 1st- Arm 2: ABX q3wk Breast Cancer line and 2nd-line MBC, Arm3: Taxol weekly with biological correlates analysis 5. ABX alone ABX:300 mg/m² q3wk Stage IIA, IIB, Phase II trial of IIIA, IIIB andneoadjuvant IV breast cancer chemotherapy (NCT) with nanoparticlepaclitaxel (ABI-007, Abraxane ®) in women with clinical stage IIA, IIB,IIIA, IIIB and IV (with intact primary) breast cancersNanoparticle Compositions

The nanoparticle compositions described herein comprise nanoparticlescomprising (in various embodiments consisting essentially of) a taxane(such as paclitaxel) and a carrier protein (such as albumin).Nanoparticles of poorly water soluble drugs (such as taxane) have beendisclosed in, for example, U.S. Pat. Nos. 5,916,596; 6,506,405; and6,537,579 and also in U.S. Pat. Pub. No. 2005/0004002A1. Although thedescription provided below is specific to taxane, it is understood thatthe same applies to other drugs, such as rapamycin, 17-AAG, and dimericthiocolchicine.

In some embodiments, the composition comprises nanoparticles with anaverage or mean diameter of no greater than about 1000 nanometers (nm),such as no greater than about any of 900, 800, 700, 600, 500, 400, 300,200, and 100 nm. In some embodiments, the average or mean diameters ofthe nanoparticles is no greater than about 200 nm. In some embodiments,the average or mean diameters of the nanoparticles is no greater thanabout 150 nm. In some embodiments, the average or mean diameters of thenanoparticles is no greater than about 100 nm. In some embodiments, theaverage or mean diameter of the nanoparticles is about 20 to about 400nm. In some embodiments, the average or mean diameter of thenanoparticles is about 40 to about 200 nm. In some embodiments, thenanoparticles are sterile-filterable.

The nanoparticles described herein may be present in a dry formulation(such as lyophilized composition) or suspended in a biocompatiblemedium. Suitable biocompatible media include, but are not limited to,water, buffered aqueous media, saline, buffered saline, optionallybuffered solutions of amino acids, optionally buffered solutions ofproteins, optionally buffered solutions of sugars, optionally bufferedsolutions of vitamins, optionally buffered solutions of syntheticpolymers, lipid-containing emulsions, and the like.

The term “proteins” refers to polypeptides or polymers of amino acids ofany length (including full length or fragments), which may be linear orbranched, comprise modified amino acids, and/or be interrupted bynon-amino acids. The term also encompasses an amino acid polymer thathas been modified naturally or by intervention; for example, disulfidebond formation, glycosylation, lipidation, acetylation, phosphorylation,or any other manipulation or modification. Also included within thisterm are, for example, polypeptides containing one or more analogs of anamino acid (including, for example, unnatural amino acids, etc.), aswell as other modifications known in the art. The proteins describedherein may be naturally occurring, i.e., obtained or derived from anatural source (such as blood), or synthesized (such as chemicallysynthesized or by synthesized by recombinant DNA techniques).

Examples of suitable carrier proteins include proteins normally found inblood or plasma, which include, but are not limited to, albumin,immunoglobulin including IgA, lipoproteins, apolipoprotein B, alpha-acidglycoprotein, beta-2-macroglobulin, thyroglobulin, transferin,fibronectin, factor VII, factor VIII, factor IX, factor X, and the like.In some embodiments, the carrier protein is non-blood protein, such ascasein, α-lactalbumin, and β-lactoglobulin. The carrier proteins mayeither be natural in origin or synthetically prepared. In someembodiments, the pharmaceutically acceptable carrier comprises albumin,such as human serum albumin. Human serum albumin (HSA) is a highlysoluble globular protein of M, 65K and consists of 585 amino acids. HSAis the most abundant protein in the plasma and accounts for 70-80% ofthe colloid osmotic pressure of human plasma. The amino acid sequence ofHSA contains a total of 17 disulphide bridges, one free thiol (Cys 34),and a single tryptophan (Trp 214). Intravenous use of HSA solution hasbeen indicated for the prevention and treatment of hypovolumic shock(see, e.g., Tullis, JAMA 237: 355-360, 460-463 (1977)) and Houser etal., Surgery, Gynecology and Obstetrics, 150: 811-816 (1980)) and inconjunction with exchange transfusion in the treatment of neonatalhyperbilirubinemia (see, e.g., Finlayson, Seminars in Thrombosis andHemostasis, 6: 85-120 (1980)). Other albumins are contemplated, such asbovine serum albumin. Use of such non-human albumins could beappropriate, for example, in the context of use of these compositions innon-human mammals, such as the veterinary (including domestic pets andagricultural context).

Human serum albumin (HSA) has multiple hydrophobic binding sites (atotal of eight for fatty acids, an endogenous ligand of HSA) and binds adiverse set of taxanes, especially neutral and negatively chargedhydrophobic compounds (Goodman et al., The Pharmacological Basis ofTherapeutics, 9^(th) ed, McGraw-Hill New York (1996)). Two high affinitybinding sites have been proposed in subdomains IIA and IIIA of HSA,which are highly elongated hydrophobic pockets with charged lysine andarginine residues near the surface which function as attachment pointsfor polar ligand features (see, e.g., Fehske et al., Biochem. Pharmcol.30: 687-92 (198a), Vorum, Dan. Med. Bull. 46: 379-99 (1999),Kragh-Hansen, Dan, Med Bull. 1441: 131-40 (1990), Curry et al., Nat.Struct. Biol. 5: 827-35 (1998), Sugio et al., Protein. Eng. 12: 439-46(1999), He et al., Nature 358: 209-15 (199b), and Carter et al., Adv.Protein. Chem. 45: 153-203 (1994)). Paclitaxel and propofol have beenshown to bind HSA (see, e.g., Paal et al., Eur. Biochem. 268(7): 2187-91(200a), Purcell et at, Biochim. Biophys. Actα1478(a): 61-8 (2000),Altmayer et al., Arzneimittelforschung 45: 1053-6 (1995), and Garrido etal., Rev. Esp. Anestestiol. Reanim. 41: 308-12 (1994)). In addition,docetaxel has been shown to bind to human plasma proteins (see, e.g.,Urien et al., Invest. New Drugs 14(b): 147-51 (1996)).

The carrier protein (such as albumin) in the composition generallyserves as a carrier for the taxane, i.e., the carrier protein in thecomposition makes the taxane more readily suspendable in an aqueousmedium or helps maintain the suspension as compared to compositions notcomprising a carrier protein. This can avoid the use of toxic solvents(or surfactants) for solubilizing the taxane, and thereby can reduce oneor more side effects of administration of the taxane into an individual(such as a human). Thus, in some embodiments, the composition describedherein is substantially free (such as free) of surfactants, such asCremophor (including Cremophor EL® (BASF)). In some embodiments, thenanoparticle composition is substantially free (such as free) ofsurfactants. A composition is “substantially free of Cremophor” or“substantially free of surfactant” if the amount of Cremophor orsurfactant in the composition is not sufficient to cause one or moreside effect(s) in an individual when the nanoparticle composition isadministered to the individual.

The amount of carrier protein in the composition described herein willvary depending on other components in the composition. In someembodiments, the composition comprises a carrier protein in an amountthat is sufficient to stabilize the taxane in an aqueous suspension, forexample, in the form of a stable colloidal suspension (such as a stablesuspension of nanoparticles). In some embodiments, the carrier proteinis in an amount that reduces the sedimentation rate of the taxane in anaqueous medium. For particle-containing compositions, the amount of thecarrier protein also depends on the size and density of nanoparticles ofthe taxane.

A taxane is “stabilized” in an aqueous suspension if it remainssuspended in an aqueous medium (such as without visible precipitation orsedimentation) for an extended period of time, such as for at leastabout any of 0.1, 0.2, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,24, 36, 48, 60, or 72 hours. The suspension is generally, but notnecessarily, suitable for administration to an individual (such ashuman). Stability of the suspension is generally (but not necessarily)evaluated at a storage temperature (such as room temperature (such as20-25° C.) or refrigerated conditions (such as 4° C.)). For example, asuspension is stable at a storage temperature if it exhibits noflocculation or particle agglomeration visible to the naked eye or whenviewed under the optical microscope at 1000 times, at about fifteenminutes after preparation of the suspension. Stability can also beevaluated under accelerated testing conditions, such as at a temperaturethat is higher than about 40° C.

In some embodiments, the carrier protein is present in an amount that issufficient to stabilize the taxane in an aqueous suspension at a certainconcentration. For example, the concentration of the taxane in thecomposition is about 0.1 to about 100 mg/ml, including for example anyof about 0.1 to about 50 mg/ml, about 0.1 to about 20 mg/ml, about 1 toabout 10 mg/ml, about 2 mg/ml to about 8 mg/ml, about 4 to about 6mg/ml, about 5 mg/ml. In some embodiments, the concentration of thetaxane is at least about any of 1.3 mg/ml, 1.5 mg/ml, 2 mg/ml, 3 mg/ml,4 mg/ml, 5 mg/ml, 6 mg/ml, 7 mg/ml, 8 mg/ml, 9 mg/ml, 10 mg/ml, 15mg/ml, 20 mg/ml, 25 mg/ml, 30 mg/ml, 40 mg/ml, and 50 mg/ml. In someembodiments, the carrier protein is present in an amount that avoids useof surfactants (such as Cremophor), so that the composition is free orsubstantially free of surfactant (such as Cremophor).

In some embodiments, the composition, in liquid form, comprises fromabout 0.1% to about 50% (w/v) (e.g. about 0.5% (w/v), about 5% (w/v),about 10% (w/v), about 15% (w/v), about 20% (w/v), about 30% (w/v),about 40% (w/v), or about 50% (w/v)) of carrier protein. In someembodiments, the composition, in liquid form, comprises about 0.5% toabout 5% (w/v) of carrier protein.

In some embodiments, the weight ratio of carrier protein, e.g., albumin,to the taxane in the nanoparticle composition is such that a sufficientamount of taxane binds to, or is transported by, the cell. While theweight ratio of carrier protein to taxane will have to be optimized fordifferent carrier protein and taxane combinations, generally the weightratio of carrier protein, e.g., albumin, to taxane (w/w) is about 0.01:1to about 100:1, about 0.02:1 to about 50:1, about 0.05:1 to about 20:1,about 0.1:1 to about 20:1, about 1:1 to about 18:1, about 2:1 to about15:1, about 3:1 to about 12:1, about 4:1 to about 10:1, about 5:1 toabout 9:1, or about 9:1. In some embodiments, the carrier protein totaxane weight ratio is about any of 18:1 or less, 15:1 or less, 14:1 orless, 13:1 or less, 12:1 or less, 11:1 or less, 10:1 or less, 9:1 orless, 8:1 or less, 7:1 or less, 6:1 or less, 5:1 or less, 4:1 or less,and 3:1 or less.

In some embodiments, the carrier protein allows the composition to beadministered to an individual (such as human) without significant sideeffects. In some embodiments, the carrier protein (such as albumin) isin an amount that is effective to reduce one or more side effects ofadministration of the taxane to a human. The term “reducing one or moreside effects of administration of the taxane” refers to reduction,alleviation, elimination, or avoidance of one or more undesirableeffects caused by the taxane, as well as side effects caused by deliveryvehicles (such as solvents that render the taxanes suitable forinjection) used to deliver the taxane. Such side effects include, forexample, myelosuppression, neurotoxicity, hypersensitivity,inflammation, venous irritation, phlebitis, pain, skin irritation,peripheral neuropathy, neutropenic fever, anaphylactic reaction, venousthrombosis, extravasation, and combinations thereof. These side effects,however, are merely exemplary and other side effects, or combination ofside effects, associated with taxanes can be reduced.

In some embodiments, the composition comprises Abraxane®. Abraxane® is aformulation of paclitaxel stabilized by human albumin USP, which can bedispersed in directly injectable physiological solution. When dispersedin a suitable aqueous medium such as 0.9% sodium chloride injection or5% dextrose injection, Abraxane® forms a stable colloidal suspension ofpaclitaxel. The mean particle size of the nanoparticles in the colloidalsuspension is about 130 nanometers. Since HSA is freely soluble inwater, Abraxane® can be reconstituted in a wide range of concentrationsranging from dilute (0.1 mg/ml paclitaxel) to concentrated (20 mg/mlpaclitaxel), including for example about 2 mg/ml to about 8 mg/ml, about5 mg/ml.

Methods of making nanoparticle compositions are known in the art. Forexample, nanoparticles containing taxanes (such as paclitaxel) andcarrier protein (such as albumin) can be prepared under conditions ofhigh shear forces (e.g., sonication, high pressure homogenization, orthe like). These methods are disclosed in, for example, U.S. Pat. Nos.5,916,596; 6,506,405; and 6,537,579 and also in U.S. Pat. Pub. No.2005/0004002A1.

Briefly, the taxane (such as docetaxel) is dissolved in an organicsolvent, and the solution can be added to a human serum albuminsolution. The mixture is subjected to high pressure homogenization. Theorganic solvent can then be removed by evaporation. The dispersionobtained can be further lyophilized. Suitable organic solvent include,for example, ketones, esters, ethers, chlorinated solvents, and othersolvents known in the art. For example, the organic solvent can bemethylene chloride and chloroform/ethanol (for example with a ratio of1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1,7:1, 8:1, or 9:a).

Other Components in the Nanoparticle Compositions

The nanoparticles described herein can be present in a composition thatinclude other agents, excipients, or stabilizers. For example, toincrease stability by increasing the negative zeta potential ofnanoparticles, certain negatively charged components may be added. Suchnegatively charged components include, but are not limited to bile saltsof bile acids consisting of glycocholic acid, cholic acid,chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid,taurochenodeoxycholic acid, litocholic acid, ursodeoxycholic acid,dehydrocholic acid and others; phospholipids including lecithin (eggyolk) based phospholipids which include the followingphosphatidylcholines: palmitoyloleoylphosphatidylcholine,palmitoyllinoleoylphosphatidylcholine,stearoyllinoleoylphosphatidylcholine stearoyloleoylphosphatidylcholine,stearoylarachidoylphosphatidylcholine, anddipalmitoylphosphatidylcholine. Other phospholipids includingL-α-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine(DOPC), distearyolphosphatidylcholine (DSPC), hydrogenated soyphosphatidylcholine (HSPC), and other related compounds. Negativelycharged surfactants or emulsifiers are also suitable as additives, e.g.,sodium cholesteryl sulfate and the like.

In some embodiments, the composition is suitable for administration to ahuman. In some embodiments, the composition is suitable foradministration to a mammal such as, in the veterinary context, domesticpets and agricultural animals. There are a wide variety of suitableformulations of the nanoparticle composition (see, e.g., U.S. Pat. Nos.5,916,596 and 6,096,331). The following formulations and methods aremerely exemplary and are in no way limiting. Formulations suitable fororal administration can consist of (a) liquid solutions, such as aneffective amount of the compound dissolved in diluents, such as water,saline, or orange juice, (b) capsules, sachets or tablets, eachcontaining a predetermined amount of the active ingredient, as solids orgranules, (c) suspensions in an appropriate liquid, and (d) suitableemulsions. Tablet forms can include one or more of lactose, mannitol,corn starch, potato starch, microcrystalline cellulose, acacia, gelatin,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, stearic acid, and other excipients, colorants, diluents,buffering agents, moistening agents, preservatives, flavoring agents,and pharmacologically compatible excipients. Lozenge forms can comprisethe active ingredient in a flavor, usually sucrose and acacia ortragacanth, as well as pastilles comprising the active ingredient in aninert base, such as gelatin and glycerin, or sucrose and acacia,emulsions, gels, and the like containing, in addition to the activeingredient, such excipients as are known in the art.

Examples of suitable carriers, excipients, and diluents include, but arenot limited to, lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, water, saline solution, syrup, methylcellulose, methyl- andpropylhydroxybenzoates, talc, magnesium stearate, and mineral oil. Theformulations can additionally include lubricating agents, wettingagents, emulsifying and suspending agents, preserving agents, sweeteningagents or flavoring agents.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation compatible with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The formulations can be presented in unit-dose or multi-dose sealedcontainers, such as ampules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Injectable formulations are preferred.

In some embodiments, the composition is formulated to have a pH range ofabout 4.5 to about 9.0, including for example pH ranges of any of about5.0 to about 8.0, about 6.5 to about 7.5, and about 6.5 to about 7.0. Insome embodiments, the pH of the composition is formulated to no lessthan about 6, including for example no less than about any of 6.5, 7, or8 (such as about 8). The composition can also be made to be isotonicwith blood by the addition of a suitable tonicity modifier, such asglycerol.

Kits

The invention also provides kits for use in the instant methods. Kits ofthe invention include a detection means for identifying the hormonereceptor status of a breast cancer patient (e.g., having tumor tissuenot expressing both estrogen receptor (ER) and progesterone receptor(PgR)). In some embodiments, the kit comprising: (a) an agent fordetecting hormone receptor status of estrogen receptor and/orprogesterone receptor of a breast cancer patient; and (b) a compositioncomprising nanoparticles comprising a taxane and a carrier protein. Insome embodiments, the kit comprising: (a) an agent for detecting hormonereceptor status of estrogen receptor and/or progesterone receptor of abreast cancer patient; and (b) instructions for assessing likelyresponsiveness to therapy for treating breast cancer based on hormonereceptor status of estrogen receptor and/or progesterone receptor,wherein the therapy comprises administering a composition comprisingnanoparticles comprising a taxane and a carrier protein. In someembodiments, the instructions further provide instructions foradministering to the patient an effective amount of the composition.

In some embodiments, the kits of the invention further include one ormore containers comprising taxane-containing nanoparticle compositions(or unit dosage forms and/or articles of manufacture). In someembodiments, the kits of the invention include one or more containerscomprising taxane-containing nanoparticle compositions (or unit dosageforms and/or articles of manufacture) and/or a chemotherapeutic agent,and in some embodiments, further comprise instructions for use inaccordance with any of the methods described herein. The kit may furthercomprise a description of selection an individual suitable or treatment.Instructions supplied in the kits of the invention are typically writteninstructions on a label or package insert (e.g., a paper sheet includedin the kit), but machine-readable instructions (e.g., instructionscarried on a magnetic or optical storage disk) are also acceptable. Insome embodiments, the instructions include instructions for treatingbreast cancer based on hormone receptor status (e.g., not expressingboth estrogen receptor (ER) and progesterone receptor (PgR)) comprisingadministering to an individual an effective amount of a compositioncomprising nanoparticles comprising a taxane and a carrier protein.

In some embodiments, the hormone receptor status is low for one or morehormone receptors such as the estrogen receptor or the progesteronereceptor. In some embodiments, the individual is likely more responsiveto the therapy if hormone receptor status is low for both estrogenreceptor and progesterone receptor. In some embodiments, the hormonereceptor status does not express (i.e., is negative for) one or morehormone receptors such as the estrogen receptor (ER) or the progesteronereceptor (PgR). In some embodiments, the hormone receptor status of thebreast cancer tissue does not express (i.e., is negative for) both theestrogen receptor (ER) and the progesterone receptor (PgR). In someembodiments, the individual is likely more responsive to the therapy ifhormone receptor status is negative for both estrogen receptor andprogesterone receptor. In some embodiments, the individual expresses(i.e., is positive for) either the estrogen receptor or the progesteronereceptor. In some embodiments, the individual expresses (i.e., ispositive for) both the estrogen receptor and the progesterone receptor.In some embodiments, the individual is likely less responsive to therapyif the hormone receptor status is positive for the estrogen receptorand/or the progesterone receptor.

In some embodiments, the breast cancer tissue further expresses HER2(HER2+). In some embodiments, the breast cancer tissue further does notexpress HER2 (HER2−).

In some embodiments, the kit comprises a) a composition comprisingnanoparticles comprising a taxane and a carrier protein (such asalbumin), b) an effective amount of at least one other chemotherapeuticagent, and c) instructions for administering the nanoparticles and thechemotherapeutic agents simultaneously and/or sequentially, fortreatment of breast cancer based on hormone receptor status (e.g., notexpressing the estrogen receptor and/or progesterone receptor). In someembodiments, the taxane is any of paclitaxel, docetaxel, and ortataxel.In some embodiments, the kit comprises nanoparticles comprising a) acomposition comprising nanoparticles comprising paclitaxel and analbumin (such as Abraxane®), b) an effective amount of at least oneother chemotherapeutic agent, and c) instructions for administering thenanoparticles and the chemotherapeutic agents simultaneously and/orsequentially, for the effective treatment of breast cancer based onhormone receptor status (e.g., not expressing the estrogen receptorand/or progesterone receptor).

In some embodiments, the kit comprises a) a composition comprisingnanoparticles comprising a taxane and a carrier protein (such asalbumin), b) a composition comprising nanoparticles comprising at leastone other chemotherapeutic agent and a carrier protein (such asalbumin), and c) instructions for administering the nanoparticlecompositions simultaneously and/or sequentially, for treatment of breastcancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor). In some embodiments,the kit comprises nanoparticles comprising a) a composition comprisingnanoparticles comprising paclitaxel and an albumin (such as Abraxane®),b) a composition comprising nanoparticles comprising at least one otherchemotherapeutic agent and a carrier protein (such as albumin), and c)instructions for administering the nanoparticle compositionssimultaneously and/or sequentially, for the effective treatment ofbreast cancer based on hormone receptor status (e.g., not expressing theestrogen receptor and/or progesterone receptor).

The nanoparticles and the chemotherapeutic agents can be present inseparate containers or in a single container. It is understood that thekit may comprise one distinct composition or two or more compositionswherein one composition comprises nanoparticles and one compositioncomprises a chemotherapeutic agent.

The kits of the invention are in suitable packaging. Suitable packaginginclude, but is not limited to, vials, bottles, jars, flexible packaging(e.g., sealed Mylar or plastic bags), and the like. Kits may optionallyprovide additional components such as buffers and interpretativeinformation.

The instructions relating to the use of the nanoparticle compositionsgenerally include information as to dosage, dosing schedule, and routeof administration for the intended treatment. The containers may be unitdoses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Forexample, kits may be provided that contain sufficient dosages of thetaxane (such as taxane) as disclosed herein to provide effectivetreatment of an individual for an extended period, such as any of aweek, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5months, 7 months, 8 months, 9 months, or more. Kits may also includemultiple unit doses of the taxane and pharmaceutical compositions andinstructions for use and packaged in quantities sufficient for storageand use in pharmacies, for example, hospital pharmacies and compoundingpharmacies.

Those skilled in the art will recognize that several variations arepossible within the scope and spirit of this invention. The inventionwill now be described in greater detail by reference to the followingnon-limiting examples. The following examples further illustrate theinvention but, of course, should not be construed as in any way limitingits scope.

EXAMPLES Example 1 Phase II Trial of Neoadjuvant Chemotherapy withAbraxane® Followed by 5-Fluorouracil, Epirubicin, and Cyclophosphamide(FEC) in Locally Advanced Breast Cancer

Abraxane® has greater efficacy and favorable toxicity compared withCremophor-based paclitaxel on a every 3 weeks schedule (Gradishar W J,et al. (2005) J Clin Oncol 23: 7794-7803). Weekly administration ofAbraxane® has shown less toxicity than the every 3 weeks schedule andactivity in taxane-refractory metastatic breast cancer (Blum J L, et al.(2004) J Clin Oncol 22: 14S, abstract 543). This trial was set up todetermine the activity and safety profile of Abraxane® followed by5-fluorouracil, Epirubicin, and cyclophosphamide (FEC) in women withlocally advanced breast cancer (LABC).

66 women with LABC were administered preoperative Abraxane® at 100 mg/m²weekly for 12 consecutive weeks, followed by FEC every 3 weeks for 4cycles. If their breast cancer was HER2 negative (HER2−) the FEC wasadministered at a dosage of 5-fluorouracil: 500 mg/m², Epirubicin: 100mg/m², Cyclophosphamide: 500 mg/m²) and referred to as FEC-100. If theircancer was HER2 positive (HER2+) the FEC was administered at a dosage of5-fluorouracil: 500 mg/m², Epirubicin: 75 mg/m², Cyclophosphamide: 500mg/m² (referred to as FEC-75) and they received trastuzumab(Herceptin®). Trastuzumab was co-administered with Abraxane® and FEC-75on a standard weekly schedule at the discretion of the investigator inpatients with HER2+ disease.

The primary endpoint of the trial was pathologic complete response ratefollowing completion of the FEC treatment. The secondary endpointsincluded complete clinical response rates assessed at the completion ofAbraxane®, toxicity/safety, progression-free survival, and overallsurvival.

The median age of the patients was 47 years (range of 28-70). Stage HBdisease was present in 33% of the patients, stage IIIA in 42%, and stageIIIB in 24%. Tumors were evaluated for hormone receptor status,including estrogen receptor (ER) and progesterone receptor (PgR). 58% ofpatients were either ER or PgR positive, and 42% were negative for bothreceptors. HER2 status was evaluated in the patients, 29% were HER2+fand 71% were HER2 negative. The protocol was completed in 58 (89.2%) ofthe patients, with therapy not completed for the rest. All 12 doses ofAbraxane were administered to 61 of the patients; 48 received the 12doses in 12 weeks, and 13 received the 12 doses in 13-14 weeks. Toxicitydata with Abraxane showed the most frequent toxicities were diarrhea,rash, fatigue, nausea and sensory neuropathy. No Grade 4 or 5 toxicitiesand only one Grade 3 neutropenia were been reported.

Abraxane at 100 mg/m² administered weekly for 12 weeks had minimaltoxicity and substantial activity, achieving a clinical completeresponse rate of 32% in patients (21/65) with locally advanced breastcancer (LABC). The sequential regimen of Abraxane followed by FEC waswell tolerated, allowing for resection in all patients andbreast-conserving surgery in 33%.

As shown in Table 3, the pathologic complete response (pCR) breast ratein HER2 positive LABC was 56% with concurrent treatment withtrastuzumab. As shown in Table 4, the pathologic complete response (pCR)breast rate in HER2 negative, hormone receptor negative LABC was 29%(5/17); the pCR breast rate in HER2 negative, HR positive LABC was 10%(3/29).

TABLE 3 HER2 STATUS pCR N = 64 N (%) Positive (18) 10 (56%) Trastuzumab7 10 No trastuzumab (1)  0 Negative (46)  8 (17%)

TABLE 4 HORMONE RECEPTOR STATUS pCR N = 64 N (%) ER and/or PgR positive(38)  7 (18%) HER2 positive (9)  4 (44%) HER2 negative (29)  3 (10%) ERand PgR negative (26) 11 (42%) HER2 positive (9)  6 (67%) HER2 negative(17)  5 (29%)

Treatment with Abraxane followed by FEC demonstrated high efficacy inboth HER2-negative patients and HER2-positive patients concurrentlytreated with trastuzumab. This treatment regimen was significantly moreeffective in patients that were ER and PgR negative than in patientsthat were ER and/or PgR positive, in both HER2-negative patients andHER2-positive patient populations.

Example 2 Improved Response and Reduced Toxicities for Abraxane®Compared to Taxol® in a Phase III Study of Abraxane® Given Every ThreeWeeks

Significantly reduced incidence of neutropenia and hypersensitivity,absence of requirement of steroid premedication, shorter duration ofneuropathy, shorter infusion time and higher dose.

ABI-007 (Abraxane®), the first biologically interactive albumin-boundpaclitaxel in a nanoparticle form, free of any solvent, was comparedwith Cremophor®-based paclitaxel (Taxol®) in individuals with metastaticbreast cancer (MBC). This phase III study was performed to confirm thepreclinical studies demonstrating superior efficacy and reduced toxicityof ABI-007 when compared with Taxol®. Individuals were randomly assignedto 3-week cycles of either ABI-007 260 mg/m² (iv) over 30 minuteswithout premedication (n=229) or Taxol® 175 mg/m² IV over 3 hours withpremedication (n=225). ABI-007 demonstrated significantly higherresponse rates compared with Taxol® (33% vs. 19%; p=0.001) andsignificantly longer time to tumor progression (23.0 vs. 16.9 weeks;HR=0.75; p=0.006). There was a trend for longer overall survival inindividuals who received ABI-007 (65.0 vs. 55.7 weeks; p=0.374). In anunplanned analysis, ABI-007 improved survival in individuals receivingtreatment as second- or greater-line therapy (56.4 vs. 46.7 weeks;HR=0.73; p=0.024). The incidence of grade 4 neutropenia wassignificantly lower in the ABI-007 group (9% vs. 22%; p<0.001) despite a49% higher paclitaxel dose. Grade 3 sensory neuropathy was more commonin the ABI-007 group than in the Taxol® group (10% vs. 2%; p<0.001) butwas easily managed and improved more rapidly (median, 22 days) than forTaxol® (median 73 days). No severe (grade 3 or 4) treatment-relatedhypersensitivity reactions occurred in any of the individuals in theABI-007 group despite the absence of premedication and shorteradministration time. In contrast, grade 3 hypersensitivity reactionsoccurred in the Taxol® group despite standard premedication (chest pain:2 individuals; allergic reaction: 3 individuals). Per protocol,corticosteroids and antihistamines were not administered routinely toindividuals in the ABI-007 group; however, premedication wasadministered for emesis, myalgia/arthralgia, or anorexia in 18individuals (8%) in the ABI-007 group in 2% of the treatment cycles,whereas 224 individuals (>99%) in the Taxol® group receivedpremedication at 95% of the cycles. The only clinical chemistry valuethat was notably different between the 2 treatment arms was higher serumglucose levels in the Taxol®-treated individuals, who also had a higherincidence of hyperglycemia reported as an AE (adverse effects) (15 [7%]vs. 3 [1%]; p=0.003). Overall, ABI-007 demonstrated greater efficacy anda favorable safety profile compared with Taxol® in this individualpopulation. The improved therapeutic index and elimination of thesteroid premedication required for solvent-based taxanes make thisnanoparticle albumin-bound paclitaxel an important advance in thetreatment of MBC.

Example 3 Weekly Abraxane® in Taxane-Refractory Metastatic Breast CancerIndividuals

A recent Phase II clinical study showed that weekly administration ofAbraxane® (nanoparticle albumin-bound paclitaxel) at a dose of 125 mg/m²resulted in long-term disease control in individuals with metastaticbreast cancer whose disease had progressed while being treated withTaxol® or Taxotere® (that is, individuals who are taxane-refractory).

Abraxane® is believed to represent the first biologically interactivecomposition that exploits the receptor-mediated (gp60) pathway found tobe integral to achieving high intracellular tumor concentrations of theactive ingredient—paclitaxel. The Phase II study included 75 individualswith taxane-refractory metastatic breast cancer. Abraxane® wasadministered weekly via a 30-minute infusion at 125 mg/m² withoutsteroid/antihistamine premedication or G-CSF prophylaxis. Individualsreceived three weekly doses followed by one week of rest, repeated every28 days. Unlike Taxol® or Taxotere®, which contain detergents that mayinhibit tumor uptake, the mechanism of action of the albumin-boundnanoparticle paclitaxel may result in improved outcomes, especially inthis difficult-to-treat individual population.

Specifically, the data showed that despite this high weekly dose of 125mg/m² in this highly pre-treated and prior taxane-exposed individualpopulation, only 3 of 75 individuals (4%) had to discontinue Abraxane®due to peripheral neuropathy. Furthermore, of those who experiencedGrade 3 peripheral neuropathy, 80% were typically able to resumetreatment after a delay of only 1 or 2 weeks and continued to receiveAbraxane® at a reduced dose for an average of 4 additional months. Thisrapid improvement was consistent with our observation from the Phase IIItrial—that the peripheral neuropathy induced by paclitaxel alone (i.e.,without Cremophor®) improves rapidly as compared to that induced byTaxol®. These Abraxane® clinical trial experiences provide the firstclinical opportunity to evaluate the effects of the chemotherapeuticagent itself, paclitaxel, from the effects from those of solvents. Basedupon both the Phase II and III experience, the data now suggest that theperipheral neuropathy from Abraxane® is not comparable to the peripheralneuropathy from Taxol® or Taxotere® with respect to duration and impacton the individual.

With regard to the clinical experience of peripheral neuropathyfollowing Taxol® or Taxotere®, Abraxis Oncology recently completed asurvey of 200 oncologists who were asked how long they thought theperipheral neuropathy induced by Taxol® took to improve and/or resolve:25% reported “7-12 months” and another 23% reported “never resolved”;for Taxotere®, the respective percentages were 29% and 7%. These dataare consistent with the statements in the Taxotere® and Taxol® packageinserts.

Analysis of the Phase II data demonstrates Abraxane® to be active inthis poor-prognosis individual population (87% visceral (lung and liver)disease, 69%>3 metastatic sites, 88% tumor growth while on taxanes), oftaxane-refractory individuals with metastatic breast cancer.Observations included a 44% disease control in Taxotere®-refractoryindividuals and 39% disease control in Taxol'-refractory individuals. Ofthose individuals whose disease progressed while on Taxotere® alone inthe metastatic setting (n=27) a 19% response rate was noted afterreceiving weekly Abraxane®. Of those individuals whose diseaseprogressed while on Taxol® alone in the metastatic setting (n=23) a 13%response rate was noted after receiving weekly Abraxane®.

Abraxane® was found to be well tolerated when administered weekly over30 minutes without steroids or G-CSF prophylaxis: Grade 4 neutropenia=3%(without G-CSF); Grade 4 anemia=1%; no severe hypersensitivity reactions(despite absence of premedication). In this heavily pretreatedindividual population, 75% of individuals were treated at the full highdose of 125 mg/m² weekly Abraxane®, with no dose reductions due totoxicities/adverse events. Of the individuals who developed grade 3sensory neuropathy, 77% were able to restart Abraxane® at a reduced dose(75-100 mg/m²) and received a mean of 12.2 (range, 1-28) additionaldoses of Abraxane®. It was remarkable to note that of these individualswho resumed Abraxane®, 80% (8 of 10) were able to restart the drugwithin 14 days after improvement of neuropathy to Grade 1 or 2. Theseresults support the observations in the pivotal Phase III trial of 260mg/m² Abraxane® administered every 3 weeks, in which rapid improvementof neuropathy (median of 22 days) was also noted. Taken together thesetwo clinical trials suggest when paclitaxel is given alone, theneuropathy which occurs appears to be short-lived and is easily managed.

Abraxane® utilizes the gp60 receptor based pathway on the microvesselendothelial cells to transport the albumin-paclitaxel complex out of theblood vessel and into the tumor interstitium, and it has been shown thatTaxol® was not transported by this mechanism. Furthermore, analbumin-binding protein, SPARC, was over-expressed in breast tumors andmay play a role in the increased intra-tumoral accumulation ofAbraxane®. The proposed mechanism suggested that once in the tumorinterstitium, the albumin-paclitaxel complex would bind to SPARC thatwas present on the tumor cell surface and be rapidly internalized intothe tumor cell by a non-lysosomal mechanism.

In addition, the surfactants/solvents commonly used in current taxaneformulations such as Cremophor®, Tween® 80 and TPGS, strongly inhibitthe binding of paclitaxel to albumin, thereby limiting transendothelialtransport. Additional data presented showed a statistically improvedefficacy of Abraxane® over Taxotere® in the MX-1 mammary breastcarcinoma xenograft at equal dose.

In conclusion, 75% of individuals were treated at fill high dose with nodose reductions. Data indicate rapid improvement of peripheralneuropathy when nanoparticle albumin-bound paclitaxel is administeredalone, without the solvent Cremophor®. Additional data provide increasedevidence that mechanism of action may play important role in enhancingindividual outcomes.

Example 4 Abraxane® (ABI-007) Acts Synergistically with TargetedAntiangiogenic Pro-Apoptotic Peptides (HKP) in MBA-MB-435 Human TumorXenografts

The antiangiogenic activity of small synthetic pro-apoptotic peptidescomposed of two functional domains, one targeting the CD13 receptors(aminopeptidase N) on tumor microvessels and the other disrupting themitochondrial membrane following internalization have previously beenreported. See Nat Med. 1999 September; 5(9): 1032-8. A second generationdimeric peptide, CNGRC-GG-d(KLAKLAK)₂, named HKP (Hunter Killer Peptide)was found to have improved antitumor activity. Since anti-angiogenicagents such as Avastin® exhibit synergism in combination with cytotoxicagents such as 5-fluorouracil, we evaluated the combination of theantiangiogenic HKP with Abraxane® (ABI-007), an albumin nanoparticlepaclitaxel that is transported by the gp60 receptor in vascularendothelium (Desai, SABCS 2003), in MDA-MB-435 human breast tumorxenografts.

Methods: MDA-MB-435 human tumor xenografts were established at anaverage tumor volume of 100 mm³, mice were randomized into groups of12-13 animals and treated with HKP, Abraxane®, or HKP and Abraxane®. HKPwas delivered i.v. (250 ug), once a week, for 16 weeks. Abraxane® wasadministered i.v., daily for 5 days at 10 mg/kg/day only for the firstweek of treatment. The Abraxane® dose used was substantially below itsMTD (30 mg/kg/day, qd×5) to prevent the tumor from complete regressionso effect of HKP could be noted.

Results: At nineteen weeks of treatment, tumor volume was significantlydecreased between control group (10,298 mm³±2,570) and HKP (4,372mm³±2,470; p<0.05 vs control) or ABI-007 (3,909 mm³±506; p<0.01 vscontrol). The combination of ABI-007 and HKP significantly reduced thetumor volume over either monotherapy (411 mm³±386; p<0.01 vs. Abraxane®monotherapy or HKP monotherapy). The treatments were well tolerated.

Conclusion: The combination of Abraxane® (ABI-007), a nanoparticlealbumin-bound paclitaxel, with the vascular targeting anti-angiogenicdimeric peptide HKP (CNGRC-GG-d(KLAKLAK)₂) against the MDA-MB-435xenograft breast tumor showed a significant reduction in tumor volumecompared to monotherapy of either agent alone. Our results suggest thatthe combination of Abraxane® with antiangiogenic agents such as HKPs orperhaps Avastin® may be beneficial.

Example 5 Metronomic ABI-007 Therapy: Antiangiogenic and AntitumorActivity of a Nanoparticle Albumin-Bound Paclitaxel Example 5a

Methods: The antiangiogenic activity of ABI-007 was assessed by the rataortic ring, human umbilical vein endothelial cell (HUVEC) proliferationand tube formation assays. Optimal dose of ABI-007 for metronomictherapy was determined by measuring the levels of circulatingendothelial progenitors (CEPs) in peripheral blood of Balb/c non-tumorbearing mice (n=5/group; dosing: 1-30 mg/kg, i.p, qd×7) with flowcytometry (Shaked et al., Cancer Cell 7: 101-111 (2005)). Subsequently,the antitumor effects of metronomic (qd; i.p.) and MTD (qd×5, 1 cycle;i.v.) ABI-007 and Taxol® were evaluated and compared in SCID micebearing human MDA-MD-231 breast and PC3 prostate cancer xenografts.

Results: ABI-007 at 5 nM significantly (p<0.05) inhibited rat aorticmicrovessel outgrowth, human endothelial cell proliferation and tubeformation by 53%, 24%, and 75%, respectively. The optimal dose ofABI-007 for metronomic therapy was observed to be 6-10 mg/kg based onCEP measurements. Metronomic ABI-007 (6 mg/kg) but not Taxol® (1.3mg/kg) significantly (p<0.05) suppressed tumor growth in both xenograftmodels. Neither ABI-007 nor Taxol® administered metronomically inducedany weight loss. Although MTD ABI-007 (30 mg/kg) inhibited tumor growthmore effectively than MTD Taxol® (13 mg/kg), significant weight loss wasnoted with the former. Interestingly, the antitumor effect of metronomicABI-007 approximated that of MTD Taxol®.

Conclusion: ABI-007 exhibits potent antiangiogenic and antitumoractivity when used in a metronomic regime.

Example 5b

Rat Aortic Ring Assay. Twelve-well tissue culture plates were coatedwith Matrigel (Collaborative Biomedical Products, Bedford, Mass.) andallowed to gel for 30 min at 37° C. and 5% CO₂. Thoracic aortas wereexcised from 8- to 10-week-old male Sprague-Dawley rats, cut into1-mm-long cross-sections, placed on Matrigel-coated wells and coveredwith an additional Matrigel. After the second layer of Matrigel had set,the rings were covered with EGM-II and incubated overnight at 37° C. and5% CO₂. EGM-II consists of endothelial cell basal medium (EBM-II;Cambrex, Walkersville, Md.) plus endothelial cell growth factorsprovided as the EGM-II Bulletkit (Cambrex). The culture medium wassubsequently changed to EBM-II supplemented with 2% FBS, 0.25 μg/mlamphotericin B and 10 μg/ml gentamycin. Aortic rings were treated withEBM-II containing the vehicle (0.9% saline/albumin),carboxyamidatriazole (CAI; 12 μg/ml), or ABI-007 (0.05-10 nM paclitaxel)for 4 days and photographed on the fifth day. CAI, a knownanti-angiogenic agent, was used at a higher than clinically achievableconcentration as a positive control. Experiments were repeated fourtimes using aortas from four different rats. The area of angiogenicsprouting, reported in square pixels, was quantified using AdobePhotoshop 6.0.

As shown in FIG. 1A, ABI-007 significantly inhibited rat aorticmicrovessel outgrowth in a concentration-dependent manner relative tothe vehicle control, reaching statistical significance (p<0.05) at 5 nM(53% inhibition) and 10 nM (68% inhibition). The amount of albuminpresent at each concentration of ABI-007 alone did not inhibitangiogenesis.

Endothelial Cell Proliferation Assay. Human umbilical vein endothelialcells (HUVEC; Cambrex) were maintained in EGM-II at 37° C. and 5% CO2.HUVECs were seeded onto 12-well plates at a density of 30,000 cells/welland allowed to attach overnight. The culture medium was then aspirated,and fresh culture medium containing either the vehicle (0.9%saline/albumin), or ABI-007 (0.05-10 nM paclitaxel) was added to eachwell After 48 h, cells were trypsinized and counted with a Coulter Z1counter (Coulter Corp., Hialeah, Fla.). All experiments were repeatedthree times.

As shown in FIG. 1B, human endothelial cell proliferation wassignificantly inhibited by ABI-007 at 5 nM and 10 nM by 36% and 41%,respectively.

Endothelial Cell Tube Formation Assay. Eight-well slide chambers werecoated with Matrigel and allowed to gel at 37° C. and 5% CO₂ for 30 min.HUVECs were then seeded at 30,000 cells/well in EGM-II containing eitherthe vehicle (0.9% saline/albumin) or ABI-007 (0.05-10 nM paclitaxel) andincubated at 37° C. and 5% CO₂ for 16 h. After incubation, slides werewashed in PBS, fixed in 100% methanol for 10 s, and stained withDiffQuick solution II (Dade Behring Inc., Newark, Del.) for 2 min. Toanalyze tube formation, each well was digitally photographed using a2.5× objective. A threshold level was set to mask the stained tubes. Thecorresponding area was measured as the number of pixels using MetaMorphsoftware (Universal Imaging, Downingtown, Pa.). Experiments wererepeated three times.

As shown in FIG. 1C, ABI-007 blocked tube formation by 75% at both 5 nMand 10 nM.

Determination of the In Vivo Optimal Biologic Dose of ABI-007 byMeasuring Circulating Endothelial Cells (CECs) and CirculatingEndothelial Progenitors (CEPs). Six- to eight-week-old female Balb/cJmice were randomized into the following eight groups (n=5 each):untreated, treated with i.p. bolus injections of either the drug vehicle(0.9% saline/albumin), or ABI-007 at 1, 3, 6, 10, 15 or 30 mg/kgpaclitaxel daily for 7 days. At the end of the treatment period, bloodsamples were drawn by cardiac puncture and collected in EDTA-containingvacutainer tubes (Becton Dickinson, Franklin Lakes, N.J.). CECs and CEPswere enumerated using four-color flow cytometry. Monoclonal antibodiesspecific for CD45 were used to exclude CD45+ hematopoietic cells. CECsand their CEP subset were depicted using the murine endothelial markersfetal liver kinase 1/VEGF receptor 2 (flk-1/VEGFR2), CD13, and CD117 (BDPharmingen, San Diego, Calif.). Nuclear staining (Procount; BDBiosciences, San Jose, Calif.) was performed to exclude the possibilityof platelets or cellular debris interfering with the accuracy of CEC andCEP enumeration. After red cell lysis, cell suspensions were evaluatedby a FACSCalibur (BD Biosciences) using analysis gates designed toexclude dead cells, platelets, and debris. At least 100,000events/sample were obtained in order to analyze the percentage of CECsand CEPs. The absolute number of CECs and CEPs was then calculated asthe percentage of the events collected in the CEC and CEP enumerationgates multiplied by the total white cell count. Percentages of stainedcells were determined and compared to the appropriate negative controls.Positive staining was defined as being greater than non-specificbackground staining. 7-aminoactinomycin D (7AAD) was used to enumerateviable versus apoptotic and dead cells.

FIG. 2 shows that ABI-007 administered i.p. daily for 7 days at 3, 10-30mg/kg significantly decreased CEP levels in non-tumor bearing Balb/cJmice. However, ABI-007 at 10-30 mg/kg was associated with a significantreduction of white blood cell count indicative of toxicity. Although thereduction of CEP levels by ABI-007 at 6 mg/kg did not reach statisticalsignificance, decrease in white blood cell count was not evident.Therefore it was concluded that the in vivo optimal biologic dose formetronomic ABI-007 was between 3-10 mg/kg. In one study, metronomicTaxol® at 1.3, 3, 6, or 13 mg/kg given i.p. daily for 7 days did notsignificantly reduce viable CEP levels, whereas metronomic Taxol® at 30mg/kg or higher resulted in severe toxicity and eventually mortality inmice. It was previously reported that the i.p. administration of Taxol®at doses commonly used in the clinic resulted in entrapment ofpaclitaxel in Cremophor® EL micelles in the peritoneal cavity andconsequently, insignificant plasma paclitaxel concentration (Gelderblomet al., Clin. Cancer Res. 8:1237-41 (2002)). This would explain whydoses of metronomic Taxol® (1.3, 3, 6, and 13 mg/kg) that did not causedeath failed to change viable CEP levels. In this case, the i.p.administration of metronomic Taxol® at 1.3 mg/kg would not be anydifferent from that at 13 mg/kg. Therefore the lower dose, 1.3 mg/kg,was selected to minimize the amount of Cremophor® EL per paclitaxeladministration for subsequent experiments.

Antitumor effects of metronomic and MTD ABI-007 compared with metronomicand MTD Taxol®. Human prostate cancer cell line PC3 and human breastcancer cell line MDA-MD-231 were obtained from the American Type CultureCollection (Manassas, Va.). PC3 cells (5×10⁶) were injected s.c. into 6-to 8-week-old male SCID mice, whereas MDA-MB-231 cells (2×10⁶) wereimplanted orthotopically into the mammary fat pad of female SCID mice.When the primary tumor volume reached approximately 150-200 mm³, animalswere randomized into eight groups (n=5-10/group). Each group was treatedwith either 0.9% saline/albumin vehicle control, Cremophor® EL vehiclecontrol, metronomic Taxol® (1.3 mg/kg, i.p., qd), metronomic ABI-007 (3,6, or 10 mg/kg paclitaxel, i.p., qd), MTD Taxol® (13 mg/kg, i.p., qd×5,1 cycle), or MTD ABI-007 (30 mg/kg paclitaxel, i.v., qd×5, 1 cycle).Perpendicular tumor diameters were measured with a caliper once a weekand their volumes were calculated. At the end of the treatment period,blood samples were drawn by cardiac puncture from mice in all groups.CECs and CEPs were enumerated as described herein.

Metronornic ABI-007 (3, 6 and 10 mg/kg) but not Taxol® (1.3 mg/kg)administered i.p. daily for 4 weeks significantly (p<0.05) inhibitedgrowth of both MDA-MB-231 and PC3 tumors (FIG. 3A and FIG. 3B). NeitherABI-007 nor Taxol® administered metronomically induced any weight loss(FIG. 3C and FIG. 3D). Although MTD ABI-007 (30 mg/kg) inhibited tumorgrowth more effectively than MTD Taxol® (13 mg/kg), significant weightloss was noted with the former, indicating toxicity. In addition, twoout of five mice treated with MTD ABI-007 displayed signs of paralysisin one limb 6 days after the last dose of drug. The paralysis wastransient and resolved within 24-48 hours. Interestingly, the antitumoreffect of metronomic ABI-007 at 6 mg/kg approximated that of MTD Taxol®in the MDA-MB-231 xenograft model (FIG. 3A). Increasing the dose ofmetronomic ABI-007 to 10 mg/kg did not seem to confer more pronouncedtumor growth inhibition. In contrast, metronomic ABI-007 elicitedgreater antitumor response at 10 mg/kg than at 3 and 6 mg/kg in the PC3xenografts (FIG. 3B).

Metronomic ABI-007 significantly decreased the levels of viable CEPs ina dose-dependent manner in MDA-MB-231 tumor-bearing mice (FIG. 4A).Viable CEP levels also exhibited a dose-dependent reduction in responseto metronomic ABI-007 in PC3 tumor-bearing mice, but reached statisticalsignificance only at 10 mg/kg (FIG. 4B). The levels of CEPs were notaltered by metronomic Taxol® in both xenograft models (FIGS. 4A and 4B).

Effects of metronomic and MTD ABI-007 and metronomic and MTD Taxol® onintratumoral microvessel density were studied. Five-um thick sectionsobtained from frozen MDA-MB-231 and PC3 tumors were stained with H&E forhistological examination by standard methods known to one skilled in theart. For detection of microvessels, sections were stained with a ratanti-mouse CD31/PECAM-1 antibody (1:1000, BD Pharmingen) followed by aTexas Red-conjugated goat anti-rat secondary antibody (1:200, JacksonImmunoResearch Laboratories, Inc., West Grove, Pa.). A singlemicrovessel was defined as a discrete cluster or single cell stainedpositive for CD31/PECAM-1d, and the presence of a lumen was not requiredfor scoring as a microvessel. The MVD for each tumor was expressed asthe average count of the three most densely stained fields identifiedwith a 20× objective on a Zeiss AxioVision 3.0 fluorescence microscopicimaging system. Four to five different tumors per each vehicle controlor treatment group were analyzed.

In MDA-MB-231 tumors, metronomic ABI-007 at 6 and 10 mg/kg as well asMTD ABI-007 seemed to reduce microvessel density (MVD) slightly althoughstatistical significance was not reached (FIG. 5A). In PC3 tumors,metronomic ABI-007 at 3 and 10 mg/kg appeared to decrease MVD butwithout reaching statistical significance (FIG. 5A). Interestingly, asignificant correlation existed between MVD and the level of viable CEPsin the MDA-MB-231 (FIG. 5B; r=0.76, P-0.04) but not in the PC3 (FIG. 5C;r=−0.071, P-0.88) model.

In vivo angiogenesis evaluation were carried out. A Matrigel plugperfusion assay was performed with minor modifications to methods knownby one skilled in the art. Briefly, 0.5 ml Matrigel supplemented with500 ng/ml of basic fibroblast growth factor (bFGF; R&D Systems Inc.,Minneapolis, Minn.) was injected s.c. on day 0 into the flanks of10-week-old female Balb/cJ mice. On day 3, animals were randomlyassigned to eight groups (n=5 each). Each group was treated with either0.9% saline/albumin vehicle control, Cremophor® EL vehicle control,metronomic Taxol® (1.3 mg/kg, i.p., qd), metronomic ABI-007 (3, 6, or 10mg/kg paclitaxel, i.p., qd), MTD Taxol® (13 mg/kg, i.v., qd×5), or MTDABT-007 (30 mg/kg paclitaxel, i.v, qd×5). As a negative control, fiveadditional female Balb/cJ mice of similar age were injected withMatrigel alone. On day 10, all animals were injected i.v. with 0.2 ml of25 mg/ml FITC-dextran (Sigma, St. Louis, Mo.). Plasma samples weresubsequently collected. Matrigel plugs were removed, incubated withDispase (Collaborative Biomedical Products, Bedford, Mass.) overnight at37° C., and then homogenized. Fluorescence readings were obtained usinga FL600 fluorescence plate reader (Biotech Instruments, Winooski, Vt.).Angiogenic response was expressed as the ratio of Matrigel plugfluorescence to plasma fluorescence.

Metronomic ABI-007 at 6 and 10 mg/kg appeared to decrease angiogenesisalthough the inhibition did not reach statistical significance (FIG. 6).Angiogenesis seemed to be unaltered by metronomic ABI-007 at 3 mg/kg,MTD ABI-007, MTD and metronomic Taxol® relative to the respectivevehicle controls (FIG. 6). These observations were similar to theintraturnoral MVD results described herein.

Example 6 Abraxane® vs Taxotere®: A Preclinical Comparison of Toxicityand Efficacy

Methods: Toxicity of Abraxane® and Taxotere® was compared in adose-ranging study in nude mice given the drugs on a q4d×3 schedule. Thedose levels were Taxotere® 7, 15, 22, 33, and 50 mg/kg and ABX 15, 30,60, 120, and 240 mg/kg. Antitumor activity of Abraxane® and Taxotere®was compared in nude mice with human MX-1 mammary xenografts at a doseof 15 mg/kg weekly for 3 weeks.

Results: In the dose-escalation study in mice, the Taxotere® maximumtolerated dose (MTD) was 15 mg/kg and lethal dose (LD₁₀₀) was 50 mg/kg.In contrast, the Abraxane® MTD was between 120 and 240 mg/kg and LD₁₀₀was 240 mg/kg. In the tumor study Abraxane® was more effective thanequal doses of Taxotere® in tumor growth inhibition (79.8% vs 29.1%,p<0.0001, ANOVA).

Conclusion: Nanoparticle abuinin-bound paclitaxel (Abraxane®) wassuperior to Taxotere® in the MX-1 tumor model when tested at equaldoses. Furthermore, the toxicity of Abraxane® was significantly lowerthan that of Taxotere®, which would allow dosing of Abraxane® atsubstantially higher levels. These results are similar to the enhancedtherapeutic index seen with Abraxane® compared to Taxol® and suggestthat the presence of surfactants may impair the transport, antitumoractivity and increase the toxicity of taxanes. Studies in additionaltumor models comparing Abraxane® and Taxotere® are ongoing.

Example 7 Combination Studies of Abraxane® and Other Agents

Due to the advantageous properties of Abraxane® (ABX, the nanoparticlealbumin-bound paclitaxel) noted above, it was used and being used in anumber of studies with different modes of administration and schedulesand in combination with other oncology drugs as well as radiationtreatment. These are listed below:

In metastatic breast cancer, these studies include:

Randomized Phase II Trial of ABX 125, Gem 1000 mg/m², To evaluate thecombination of ABX Weekly Abraxane ® in Combination D1, 8; q 3wk andGemcitabine in 1st-line MBC. with Gemcitabine in Individuals with HER2Negative Metastatic Breast Cancer A phase II study of weekly ABX 100mg/m², Carbo AUC Data will be important for using dose-densenanoparticle paclitaxel 2, both D1, 8, 15; Her 2 mg/kg ABX incombination with carbo (ABI-007) carboplatin, with (4 mg/kg on wk a)q4wk × 6 and/or Herceptin ®. Also helpful for Herceptin ® as first orsecond-line other combinations. therapy of advanced HER2 positive breastcancer Weekly Vinorelbine and Abraxane ®, L1: ABX 80, Nav 15; L2:Multi-center study of ABX in with or without G-CSF, in stage IV ABX 90,Nav 20; L3: ABX combination with Navelbine ® in breast cancer: a phaseI-II study 100, Nav 22.5; L4: ABX 110, 1st-line MBC. Nav 25; L5: ABX125, Nav 25 qwk Phase II trial of weekly Abraxane ® ABX 125 mg/m² Q3/4wkA relatively large phase II of weekly monotherapy for 1st-line MBC (plusABX monotherapy at 125 mg/m² in Herceptin ® in Her2+ pts) 1st-line MBC.Phase I/II trial Abraxane ® plus ABX + Anthracycline Doxil ® for MBCplus limited PK 3-arm phase II trial in 1st-line MBC ABX weekly (130mg/m²) vs. To optimize ABX monotherapy q2wk (260 mg/m²) vs. q3wk regimefor MBC (260 mg/m²) 3-arm phase II trial in 1st-line MBC ABX weekly vs.ABX q3wk randomized ABX MBC trial to 2nd-line MBC, with biological vs.Taxol ® weekly obtain important data: weekly ABX correlates analyses vs.weekly Taxol ®; weekly ABX vs. 3-weekly ABX; plus biomarker study(caveolin-1 and SPARC), Phase I/II Abraxane ® + GW572016 TBD combinationof ABX and GW572016 (a dual EGFR inhibitor and one of the most promisingnew biological agents for BC). A phase I dose escalation study of aAbraxane ® 100 mg/m² weekly, This phase I trial is to determine the 2day oral gefitinib 3 out of 4 weeks; Gefitinib safety and tolerabilityof a 2 day chemosensitization pulse given prior starting at 1000 mg/d ×2 days gefitinib pulse given prior to to weekly Abraxane ® inindividuals Abraxane ® administration. with advanced solid tumors PhaseII 1^(st) line MBC trial weekly ABX (125 mg/m², 2 To evaluate thecombination of ABX wk on and 1 wk off) + Xeloda ® and Xeloda ® in1st-line MBC, using 825 mg/m² d 1-14 q3wk 2 weekly on and 1 weekly offABX regime. Phase II pilot adjuvant trial of Dose dense AC + G CSF --> Apilot: adjuvant study of a “super Abraxane ® in breast cancer weekly ABX--> Avastin ® dose dense” Abraxane ® in dose-dense adjuvant AC q2w × 4 +G CSF --> ABX A pilot adjuvant study of dose dense chemotherapy forearly stage breast q2wk × 4 ABX regime—an alternate of a cancer standardadjuvant regime Phase II pilot adjuvant trial of AC Q2wk --> ABX q2wk +A pilot adjuvant study in preparation Abraxane ® in breast cancer G-CSFfor phase III adjuvant trial

In Breast cancer neoadjuvant setting studies include:

Phase II Trial of Dose Dense Neoadjuvant: Gem 2000, This neoadjuvantstudy is based on the Neoadjuvant Gemcitabine, Epirubicin, Epi 60, ABX175 mg/m², GET data from Europe which showed ABI-007 (GEA) in LocallyAdvanced Neul 6 mg SC, all D1 q2 high activity. In the current regime,or Inflammatory Breast Cancer wk × 6 Adjuvant: Gem ABX will replace T,or Taxol ®. 2000, ABX 220, Neul 6 mg D1 q2wk × 4 Phase II preoperativetrial of ABX 220 mg/m2 q2wk × Abraxane ® followed by FEC 6 followed byFEC × 4 (+Herceptin ® as appropriate) in (+Herceptin ® for Her2+ breastcancer pts) Pre-clinical study of drug-drug ABX + other agentsinteraction Phase II neoadjuvant (ABX + Herceptin ®) followed by(Navelbine ® + Herceptin ®) Randomized phase II trial of TAC vs. ACfollowed To evaluate AC followed by neoadjuvant chemotherapy in ABX +carbo vs. AC ABX/carbo or ABX/carbo/Herceptin ® individuals with breastcancer followed combinations vs TAC (a FDA ABX + carbo + Herceptin ®approved adjuvant BC regime) in neoadjuvant setting. Phase IIneoadjuvant trial of ABX: 200mg/m² D1, Abraxane ® and capecitabine inXel: 1000 mg/m² D1-14; locally advanced breast cancer q3wk x 4 Phase IItrial of neoadjuvant ABX: 300 mg/m² q3wk chemotherapy (NCT) withnanoparticle paclitaxel (ABI-007, Abraxane ®) in women with clinicalstage IIA, IIB, IIIA, IIIB, and IV (with intact primary) breast cancers

Studies in Chemoradiation include:

Abraxane ® Combined animal model With Radiation

Other studies include:

Ph II single treatment use of ABI-007 for the treatment ofnon-hematologic malignancies Abraxane ® Combined With antiangiogenicagents, e.g., Avastin ®. Abraxane ® Combined With proteasome inhibitorse.g., Velcade ®. Abraxane ® Combined With EGFR inhibitors e.g.,Tarceva ®.

Example 8 Combination of Abraxane® with Carboplatin and Herceptin®

The combination of Taxol® and carboplatin has shown significant efficacyagainst metastatic breast cancer. On a weekly schedule, in thiscombination, Taxol® can only be dosed at up to 80 mg/m². Higher dosescannot be tolerated due to toxicity. In addition, HER-2-positiveindividuals derive greater benefit when Herceptin® is included in theirtherapeutic regime. This open-label Phase II study was conducted todetermine the synergistic therapeutic effect of ABI-007 (Abraxane®) withthese agents. The current study was initiated to evaluate the safety andantitumor activity of ABI-007/carboplatin with Herceptin® forindividuals with HER-2 positive disease. ABI-007 was given incombination with carboplatin and Herceptin® administered intravenouslyweekly to individuals with HER-2 positive advanced breast cancer. Acohort of 3 individuals received ABI-007 at a dose of 75 mg/m² IVfollowed by carboplatin at target AUC=2 weekly and Herceptin® infusion(4 mg/kg at week 1, and 2 mg/kg on all subsequent weeks) for 1 cycle.These individuals tolerated the drug very well so for all subsequentcycles and individuals the dose of ABI-007 was escalated to 100 mg/m².Six individuals were treated to date. Of the 4 individuals that wereevaluated for response, all 4 (100%) showed a response to the therapy.It should be noted that due to lower toxicity of Abraxane®, a highertotal paclitaxel dose could be given compared to Taxol® with resultingbenefits to the individuals.

Example 9 Combination of Abraxane® and Tyrosine Kinase Inhibitors

Pulse-dosing of gefitinib in combination with the use of Abraxane® isuseful to inhibit the proliferation of EGFR expressing tumors. 120 nudemice are inoculated with BT474 tumor cells to obtain at least 90 micebearing BT474 xenograft tumors and split into 18 experimental arms (5mice each). Arm 1 mice receive control i.v. injections. All other micereceive weekly i.v. injections of Abraxane® at 50 mg/kg for 3 weeks. Arm2 receives Abraxane® alone. Arms 3, 4, 5, 6, 7, 8 receive weeklyAbraxane® preceded by 2 days of a gefitinib pulse at increasing doses.Arms 9, 10, 11, 12, 13 receive weekly Abraxane® preceded by one day of agefitinib pulse at increasing doses. Arms 14, 15, 16, 17, 18 receiveweekly Abraxane® along with everyday administration of gefitinib atincreasing doses. The maximum tolerated dose of gefitinib that can begiven in a 1 or 2 day pulse preceding weekly Abraxane® or in continuousadministration with Abraxane® is established. In addition, measurementof anti-tumor responses will determine whether a dose-responserelationship exists and whether 2 day pulsing or 1 day pulsing issuperior. These data are used to select the optimal dose of pulsegefitinib and that of continuous daily gefitinib given with Abraxane®.

120 nude mice are inoculated with BT474 tumor cells to obtain 90 micebearing tumors. These mice are split into 6 groups (15 each). Arm 1receive control i.v. injections. Arm 2 receives Abraxane® 50 mg/kg i.v.weekly for 3 weeks. Arm 3 receive oral gefitinib at 150 mg/kg/day. Arm 4receive Abraxane® 50 mg/kg along with daily gefitinib at the previouslyestablished dose. Arm 5 receive Abraxane® 50 mg/kg preceded by agefitinib pulse at the previously established dose and duration. Arm 6receive only a weekly gefitinib pulse at the previously establisheddose. After three weeks of therapy, mice are followed until controlsreach maximum allowed tumor sizes.

Example 10 Phase II Study of Weekly, Dose-dense Nab™-Paclitaxel(Abraxane®), Carboplatin With Trastuzumab® As First-line Therapy OfAdvanced HER-2 Positive Breast Cancer

This study aimed to evaluate (1) the safety and tolerability and (2) theobjective response rate of weekly dose-densetrastuzumab/Abraxane®/carboplatin as first-line cytotoxic therapy forpatients with advanced/metastatic (Stage IV adenocarcinoma)HER-2-overexpressing breast cancer. Trastuzumab is a monoclonalantibody, also known as Herceptin®, which binds to the extracellularsegment of the erbB2 receptor.

Briefly, patients without recent cytotoxic or radiotherapy wereincluded. Doses of Abraxane® were escalated from 75 mg/m² as 30-min i.v.infusions on days 1, 8, 15 up to 100 mg/m² for subsequent cyclesaccording to the standard 3+3 rule. Carboplatin AUC=2 was given as 30-60min i.v. infusions on days 1, 8, 15 and for an initial 29 day cycle.Trastuzumab was given as i.v. 30-90 min infusion on days 1, 8, 15, 22 ata dose of 4 mg/kg at week 1 and 2 mg/kg on all subsequent weeks.

Of 8 out of 9 patients evaluable for response the response rate(confirmed plus unconfirmed) was 63% with 38% stable disease. The mostcommon toxicities were neutropenia (grade 3: 44%; grade 4: 11%) andleukocytopenia (33%).

These results suggest that trastuzumab plus Abraxane® plus carboplatindemonstrated a high degree of antitumor activity with acceptabletolerability as a first-line therapy for MBC.

Example 11 Phase II Trial of Capecitabine Plus Nab™-Paclitaxel(Abraxane®) in the First Line Treatment of Metastatic Breast Cancer

The purpose of this phase II study was to evaluate the safety, efficacy(time to progression and overall survival), and quality of life ofpatients with MBC who received capecitabine in combination withAbraxane®. Capecitabine is a fluoropyrimidine carbamate also known asXeloda® which has been shown to have substantial efficacy alone and incombination with taxanes in the treatment of MBC.

In this open-label, single-arm study, Abraxane® 125 mg/m² was given byi.v. infusion on day 1 and day 8 every 3 weeks plus capecitabine 825mg/m² given orally twice daily on days 1 to 14 every 3 weeks. Patientswere HER-2/neu negative with a life expectancy of greater than 3 months.Patients had no prior chemotherapy for metastatic disease, no priorcapecitabine therapy, and no prior fluoropyrimidine therapy andpaclitaxel chemotherapy given in an adjuvant setting. Over the course ofthe trial, 3 patients required a dose reduction of capecitabine to 650mg/m², 2 patients required a dose reduction of capecitabine to 550 mg/m²and 3 patients required a dose reduction of Abraxane® to 100 mg/m².

The primary endpoint was objective response rate and safety/toxicity,with evaluation performed after every 2 cycles. A secondary endpoint wastime to progression. 12 patients have been enrolled with safety analysiscompleted on the first 6 patients and the response rate evaluable after2 cycles in the first 8 patients. There were no unique or unexpectedtoxicities with no grade 4 toxicities or neuropathy greater thangrade 1. Response data were confirmed on only the first 2 cycles oftherapy (first evaluation point) in 6 patients. Two patients havecompleted 6 cycles with 1 partial response and 1 stable disease. Of thefirst 8 patients after 2 cycles, there were 2 partial responses and 4with stable disease.

Subsequently, a total of 50 patients were enrolled and 38 were availablefor analysis. Average age of the patients was 58.3 years (range of24-84) and 50% of the patients had prior chemotherapy treatment (priorto metastatic disease). 34% of the patients had 1 metastatic site, 37%had 2 sites, 21% had 3 sites and 8% had more than 3 sites. The mostcommon sites for metastases were the liver, bone, pulmonary tissue andother lymph nodes.

These results show that combination of capecitabine and weekly Abraxane®at effective doses is feasible with no novel toxicities to date.Abraxane® related toxicity was mainly neutropenia without clinicalconsequences, and hand foot syndrome was the major toxicity ofcapecitabine.

The clinical response was evaluated in 34 patients with 3 (9%)demonstrating complete response, 15 (44%) demonstrating partialresponse, 11 (32%) demonstrating stable disease and 5 (15%)demonstrating progressive disease. The combination of capecitabine andAbraxane was a very active combination regimen in first line metastaticbreast cancer treatment. The results demonstrated a prolonged time toprogression with this combination regimen.

Example 12 Pilot Study of Dose-Dense Doxorubicin Plus CyclophosphamideFollowed by Nab-Paclitaxel (Abraxane®) in Patients with Early-StageBreast Cancer

The objective of this study was to evaluate the toxicity of doxorubicin(adriamycin) plus cyclophosphamide followed by Abraxane® in early stagebreast cancer.

Patients had operable, histologically confirmed breast adenocarcinoma ofan early stage. The patients received doxorubicin (adriamycin) 60 mg/m²plus cyclophosphamide 600 mg/m² (AC) every 2 weeks for 4 cycles followedby Abraxane® 260 mg/m² every two weeks for 4 cycles.

30 patients received 4 cycles of AC, and 27 of 29 patients received 4cycles of Abraxane®; 33% of patients received pegfilgrastim (Neulasta®)for lack of recovery of ANC (absolute neutrophil count) duringAbraxane®. Nine patients (31%) had Abraxane® dose reductions due tonon-hematologic toxicity. A total of 9 patients had grade 2 and 4patients had grade 3 peripheral neuropathy (PN); PN improved by ≥1 gradewithin a median of 28 days.

These results indicate that dose-dense therapy with doxorubicin (60mg/m²) plus cyclophosphamide (600 mg/m²) every 2 weeks for 4 cyclesfollowed by dose-dense Abraxane® (260 mg/m²) every 2 weeks for 4 cycleswas well tolerated in patients with early-stage breast cancer.

Example 13 Weekly Nab-Paclitaxel (Abraxane®) as First Line Treatment ofMetastatic Breast Cancer with Trastuzumab Add on for HER-2/Neu-PositivePatients

The purpose of the current study was to move weekly Abraxane® to afront-line setting and add trastuzumab for HER2/neu-positive patients.

This phase II, open-label study included 20 HER2-positive and 50HER2-negative patients with locally advanced or metastatic breastcancer. Abraxane® was given at 125 mg/m² by 30 minute i.v. infusion ondays 1, 8, and 15 followed by a week of rest. Trastuzumab was givenconcurrently with study treatment for patients who were HER2-positive.The primary endpoint was response rate and the secondary endpoints weretime to progression (TIP), overall survival (OS), and toxicity.

In the safety population, 23 patients received a median of 3 cycles ofAbraxane® to date. The most common treatment-related adverse event wasgrade 3 neutropenia (8.7%) with no grade 4 adverse events. One out of 4evaluable patients responded to therapy.

Example 14 Phase I Trial of Nab-Paclitaxel (Abraxane®) and Carboplatin

The aim of the current study was to determine the maximum tolerated doseof Abraxane® (both weekly and every 3 weeks) with carboplatin AUC=6 andto compare the effects of sequence of administration on pharmacokinetics(PK).

Patients with histologically or cytologically documented malignancy thatprogressed after “standard therapy” were included. Arm 1 receivedAbraxane® every 3 weeks in a dose escalation format based on cycle 1toxicities (220, 260, 300, 340 mg/m²) every 3 weeks followed bycarboplatin AUC=6. Arm 2 received weekly (days 1, 8, 15 followed by 1week off) Abraxane® (100, 125, 150 mg/m²) followed by carboplatin AUC=6.For the PK portion of the study, Abraxane® was followed by carboplatinin cycle 1 and the order of administration reversed in cycle 2 with PKlevels determined at initial 6, 24, 48 and 72 hours.

On the every 3 weeks schedule, neutropenia, thrombocytopenia andneuropathy were the most common grade 3/4 toxicities (3/17 each). On theweekly schedule, neutropenia 5/13 was the most common grade 3/4toxicity. The best responses to weekly administration at the highestdose of 125 mg/m² (n=6) were 2 partial responses (pancreatic cancer,melanoma) and 2 stable disease (NSCLC). The best responses to the everythree week administration at the highest dose of 340 mg/m² (n=5) were 1stable disease (NSCLC) and 2 partial responses (SCLC, esophageal).

These data indicate activity of combination of Abraxane® andcarboplatin. The MTD for the weekly administration was 300 mg/m², andfor the once every 3 week administration was 100 mg/m².

Example 15 Phase II Trial of Dose-Dense Gemcitabine, Epirubicin, andNab-Paclitaxel (Abraxane®) (GEA) in Locally Advanced/Inflammatory BreastCancer

Gemcitabine, anthracyclines and taxanes are among the most active agentsin the treatment of breast cancer with metastatic breast cancer (MBC)trials confirming the high activity of this triplet. As neoadjuvanttherapy, pathologic complete response (pCR) rates of 25% are evidentwith a variety of schedules. Previous neoadjuvant trial of gemcitabine,epirubicin and weekly docetaxel demonstrated a 24% pCR following 12weeks of therapy although with prohibitive hematologic toxicity.Abraxane® is a novel taxane with superior responses, time to progression(TIP), and less myelosuppression than standard paclitaxel. Uniqueenhanced intratumoral concentrations for Abraxane® have been attributedto gp60 and SPARC glycoproteins. This trial examined the feasibility,toxicity, and efficacy of dose dense neoadjuvant combination therapy ofgemcitabine, epirubicin and Abraxane® (GEA) in locally advanced breastcancer and/or inflammatory breast cancer. Primary objectives were toassess feasibility and toxicity of neoadjuvant GEA and to evaluate theclinical and pathological responses. The secondary objectives were toassess time to progression, overall survival and rate of breastconserving surgery.

In an open-label, phase II study an induction/neoadjuvant therapy regimewas instituted prior to local intervention. The therapy regime wasgenicitabine 2000 mg/m² i.v. every 2 weeks for 6 cycles, epirubicin 50mg/m² every 2 weeks for 6 cycles, Abraxane® 175 mg/m² every 2 weeks for6 cycles, with pegfilgrastim 6 mg s.c. on day 2 every 2 weeks. Thepostoperative/adjuvant therapy regime after local intervention wasgemcitabine 2000 mg/m² every 2 weeks for 4 cycles, Abraxane® 220 mg/m²every 2 weeks for 4 cycles and pegfilgrastim 6 mg s.c. day every 2weeks. Patients included females with histologically confirmed locallyadvanced/inflammatory adenocarcinoma of the breast.

48 patients with a median age of 48 years (range 29-73) were enrolled.Tumor characteristics included 79% with ductal histology, 8% withlobular histology and 13% with inflammatory histology. Hormone receptorstatus included i) estrogen receptor (ER) negative and progesteronereceptor (PgR) negative tumors-54% of patients; ii) ER+PgR+ tumors-33%of patients; iii) ER+PR-tumors-10% of patients; and iv) ER-PR+ tumors-2%of patients. HER2 status was shown to be HER2+ tumors-81% of patientsand HER2-tumors-19% of patients. 113 cycles have been administered. 18patients have completed the post-op therapy. Hemotologic toxicityprimarily consisted of G3/4 neutropenia, 4 patients (8%); G3thrombocytopenia, 3 patients (6%); and G3 anemia, 1 patient (2%). Therewere no episodes of febrile neutropenia. Non-hematologic toxicity wasminimal with only 1 G4 event (fatigue) G3 events primarily consisted ofarthralgia/pain, 5 patients (10%); neuropathy and infection each werenoted in 2 patients.

35 patients were available to evaluate for pathological findings andcombination drug therapy efficacy. Pathologic complete response (pCR;defined as pathologic complete responses in both the primary breast andlymph node tissue) was 20% (7/35); pathologic partial response was 74%(26.35); and stable disease was 6% (2/35).

The results demonstrated that dense combination therapy at neoadjuvantdoses with gemcitabine, epirubicin and Abraxane® (GEA) was feasible andwell-tolerated. Toxicity with the biweekly schedule was minimal andeasily manageable. GEA combination therapy demonstrated high levels ofcomplete or partial response rates.

Example 16 Abraxane® (ABI-007) Reduces Tumor Growth in MDA-MB-231 HumanTumor Xenografts and Induces Necrosis, Hypoxia and VEGF-A Expression

MDA-MB-231 human breast cancer xenografts were orthotopically implantedin the mammary fat pads of female nude (nu/nu) mice. When the averagetumor volume was 230 mm³, mice were randomized into groups of fiveanimals and treated with saline, Taxol®, Abraxane® or doxorubicin.Taxol® was administered at 10 mg/kg/day, Abraxane® was administered at15 mg/kg/day, and doxorubicin was administered at 10 mg/kg/day. Alldrugs and control saline were administered i.v. in a volume of 100 μldaily for 5 days. Mice were sacrificed, tumors were harvested and tumorcellular extracts were prepared. VEGF-A protein levels in tumor extractswere determined by ELISA. In some cases, tumors from mice treated withAbraxane® were analyzed by histology.

TABLE 5 Mean VEGF-A Dose Tumor Volume (pg/mg Treatment Schedule (mm³) %TGI protein) Saline control 100 μl 523 ± 79 337 ± 51 qdx5 Taxol ® 10mg/kg/day 231 ± 32 56 664 ± 66 qdx5 Abraxane ® 15 mg/kg/day 187 ± 29 64890 ± 82 qdx5 Doxorubicin 10 mg/kg/day 287 ± 56 45 754 ± 49 qdx5

As shown in Table 5, Taxol®, Abraxane® and doxorubicin all inhibitedtumor growth as represented by a reduction in tumor volume when comparedto saline-treated control animals. Tumor growth inhibition (TGI) wascalculated by comparing mean tumor volume of test groups to that of thecontrol group at the last measurement of the control group. Tumor growthinhibition was greatest in mice treated with Abraxane® (64% inhibition).Taxol® and doxorubicin showed tumor growth inhibition of 56% and 45%,respectively.

Levels of VEGF-A protein in tumor cellular extracts were measured byELISA and shown to be increased in tumors from mice treated with Taxol®,Abraxane® and doxorubicin. VEGF-A protein levels were highest in tumorsfrom mice treated with Abraxane® (164% increase), followed bydoxorubicin (124%) and Taxol® (97%).

Tumors were harvested from saline-treated control mice andAbraxane®-treated mice one week after the last injection of Abraxane®.The tumors were evaluated for sites of necrosis and for the presence ofhypoxic cells. Hypoxic cells were identified by immunohistochemicaldetection of pimonidazole-protein conjugates. As shown in FIG. 7 FIGS.7A-7D, inhibition of tumor growth in Abraxane®-treated mice wasaccompanied by necrosis (FIG. 7B) and hypoxia (FIG. 7D) in the tumortissue. Necrosis and hypoxia was not observed in tumor tissue fromsaline-treated control mice (FIG. 7A and FIG. 7C).

Example 17 VEGF-A and Avastin® Effects on Abraxane®-Induced in VitroCytotoxicity

The effect of VEGF-A or an anti-VEGF antibody (Avastin®) onAbraxane®-induced cytotoxicity was assessed in an in vitro cellularcytotoxicity assay. Cells were treated with Abraxane® in a range ofconcentrations (1 to 24 nM). Cells were also treated with VEGF-A orAvastin® and cytotoxicity was compared to cells treated with Abraxane®alone. As shown in FIG. 8A, addition of VEGF-A reduced the in vitrocytotoxicity of Abraxane®. In contrast, the addition of Avastin®increased the in vitro cytotoxicity of Abraxane® (FIG. 8A).

Similar results were observed in an in vitro clonogenic assay. Cellswere treated with saline control, Abraxane® alone, VEGF-A alone,Avastin® alone, Abraxane®+VEGF-A or Abraxane®+Avastin®. As shown in FIG.8B, Abraxane® reduced the mean number of colonies formed as compared tosaline control. Treatment with VEGF-2 alone increased the number ofcolonies formed, while treatment with Avastin® alone resulted in aslight reduction in the number of colonies formed. The addition ofVEGF-A to Abraxane®-treated cells reduced the cytotoxic effect whichresulted in a higher number of colonies formed as compared to Abraxane®alone. The addition of Avastin® to Abraxane®-treated cells appeared tohave a synergistic effect demonstrating an increase in cytotoxicity (asdemonstrated by a sharp decrease in number of colonies formed) over thelevel observed with Abraxane® or Avastin® alone.

Example 18 Abraxane® (ABI-007) in Combination with Avastin® ReducesTumor Growth in MDA-MB-231 Tumor Xenografts

Luciferase-expressing MDA-MB-231 human breast cancer xenografts wereorthotopically implanted in the mammary fat pads of female nude (nu/nu)mice. When the average tumor volume reached 230 mm³, mice wererandomized into groups of five animals and treated with saline,Abraxane®, Avastin®, or a combination of Abraxanc® plus Avastin®.Abraxane®, either alone or in combination, was administered at 10mg/kg/day daily for 5 days in two cycles separated by 1 week. Somegroups were administered Abraxane® at 10 mg/kg daily for 5 days for onlyone cycle. Avastin® was administered following the two cycles ofAbraxane® at dosages of 2 mg/kg, 4 mg/kg or 8 mg/kg twice a week for 6weeks. Avastin® alone was administered at a dosage of 4 mg/kg at thesame time as mice in combination therapy. Mice were monitored for tumorgrowth and drug toxicity. Mice were sacrificed when the mean tumorvolume in the saline-treated control group reached 2000 mm³.

TABLE 6 Mean Avastin ® Tumor Volume % % Complete Treatment Dose (mm³)TGI Regression³ Saline control 2391 ± 432 0 Abraxane ® 117 ± 38 95.11 0(ABX) Avastin ® 4 mg/kg 2089 ± 251 12.56 0 ABX + 2 mg/kg 138 ± 42 94.2320 (1/5) Avastin ® ABX + 4 mg/kg  60 ± 17 97.49 40 (2/5) Avastin ® ABX +8 mg/kg  36 ± 16 98.49 40 (2/5) Avastin ®

No toxicity was observed in any treatment group. Tumor growth inhibition(TGI) was calculated by comparing mean tumor volume of test groups tothat of the control group at the last measurement of the control group.As shown in Table 6 and in FIG. 9, Avastin® at a dose of 4 mg/kg did notsignificantly inhibit growth of primary tumors (12.56% inhibition).Abraxane® and Avastin® combination therapy, particularly with 2 cyclesof Abraxane®, yielded a significantly better outcome than Avastin®alone, with tumor inhibition ranging from 94.23% to 98.49%. Abraxane® incombination with Avastin® at the two highest doses, yielded a betteroutcome that Abraxane® alone (97.49 or 98.49% compared to 95.11%inhibition). Abraxane® and Avastin® in combination resulted inregression of tumors in treated mice wherein complete regressionreferred to mice with no measurable tumors at day 65. Five of fifteen(30%) mice treated with a combination of Abraxane® and Avastin® showedcomplete tumor regression; tumors in the remaining mice were reduced by90% compared with controls.

At these doses, Avastin® alone did not significantly inhibit primarytumors. The efficacy of Abraxane® was much higher than that of Avastin®and was substantially improved by adding a second cycle of Abraxane®.Only the combination of Abraxane® and Avastin® eradicated primary tumorsin 30% of the mice.

Example 19 Abraxane® (ABI-007) in Combination with Avastin® ReducesTumor Metastasis in MDA-MB-231 Tumor Xenografts

As described in Example 25, luciferase-expressing MDA-MB-231 humanbreast cancer xenografts were orthotopically implanted in the mammaryfat pads of female nude (nu/nu) mice. When the average tumor volumereached 230 mm³, mice were randomized into groups and treated withsaline (n=10), Abraxane® (n=5), Avastin® (n=5), or a combination ofAbraxane® plus Avastin® (n=5). Abraxane®, either alone or incombination, was administered at 10 mg/kg/day daily for 5 days in twocycles separated by 1 week. Some groups were administered Abraxane® at10 mg/kg daily for 5 days for only one cycle. Avastin® was administeredfollowing the two cycles of Abraxane® at dosages of 2 mg/kg, 4 mg/kg or8 mg/kg twice a week for 6 weeks. Avastin® alone was administered at adosage of 4 mg/kg at the same time as mice in combination therapy. Micewere sacrificed when the mean tumor volume in the saline-treated controlgroup reached 2000 mm³. Axillary lymph nodes and both lobes of the lungswere removed from each mouse and cellular extracts were prepared. Thepresence of MDA-MB-231 cells in these tissues was evaluated by analysisof luciferase activity and was an indicator of metastasis from theprimary tumor. Luciferase activity was measured in extracts from 10lymph nodes and both lobes of the lungs on the day of sacrifice (day 65after tumor implantation). A value greater than 500 light units per 20μl lysate was rated as positive for presence of MDA-MB-231 cells and forincidence of metastasis.

TABLE 7 Lymph Node Pulmonary Metastasis Metastasis Avastin ® P PTreatment Dose Incidence Value Incidence Value Saline 10/10 (100%) 7/10(70%) control Abraxane ®  5/5 (100%) —  4/5 (80%) — (ABX) Avastin ® 4mg/kg  5/5 (100%) —  3/5 (60%) NS ABX + 2 mg/kg  5/5 (100%) —  1/5 (20%)0.045 Avastin ® ABX + 4 mg/kg  2/5 (40%) 0.022  2/5 (40%) NS Avastin ®ABX + 8 mg/kg  2/5 (40%) 0.022 0/5 (0%) 0.025 Avastin ®

As shown in Table 7, treatment with Abraxane® or Avastin® alone appearedto have no effect on the incidence of tumor metastasis to the lymphnodes as analyzed by luciferase activity in cellular extracts. As usedherein, incidence refers to the presence of luciferase activity intissue from each mouse. Abraxane® in combination with Avastin® diddemonstrate a significant effect on tumor metastasis, particularly with2 cycles of Abraxane®. Incidence of metastases fell to 40% in groupstreated with Abraxane® and Avastin® at the two highest dosages of 4mg/kg and 8 mg/kg. (P value=0.022; wherein the P value was generated bythe analysis of difference between test and control groups with Fisher'sexact test; NS refers to non-significant.) Abraxane® or Avastin® aloneappeared to have little effect on incidence of tumor metastasis to thelungs as shown in Table 6. Abraxane® in combination with Avastin®demonstrated an effect on the incidence of lung metastases. Incidence ofmetastases fell to 20%, 40% and 0% with combinations of Abraxane® andAvastin® at dosages of 2 mg/kg, 4 mg/kg and 8 mg/kg, respectively.

Tumor metastasis to the lymph nodes and lungs as evaluated by luciferaseactivity in tissue extracts is shown in FIGS. 10A and 10B. Thecombination of Abraxane® and Avastin® had a synergistic effect onreducing lymph node metastases (FIG. 10A) and lung metastases (FIG. 10B)of the MDA-MB-231 tumor cells. At these doses, Avastin® alone did notsignificantly inhibit metastasis. The efficacy of Abraxane® was muchhigher than that of Avastin® and was substantially improved by adding asecond cycle of Abraxane®. Only the combination of Abraxane® andAvastin® eliminated regional and distant metastases in a proportion ofthe treated mice.

Example 20 Abraxane® (ABI-007) in Combination with Avastin® forTreatment of Metastatic Breast Cancer

The combination of bevacizumab (Avastin®) and paclitaxel has significantactivity in metastatic breast cancer. Abraxane® is less toxic and hasdemonstrated a better tumor delivery than paclitaxel, therefore thecombination of Abraxane® and Avastin® was used to treat women withmetastatic breast cancer.

27 women with metastatic breast cancer were treated with a combinationof Abraxane® and Avastin®. Abraxane® was administered at 80-125 mg/m² onday 1, 8 and 15 (once a week for 3 weeks) or 170-200 mg/m² every 14 days(once every 2 weeks) on a 28 day cycle, Avastin® was administered at 10mg/m² every 14 days (once every 2 weeks). A minimum of two cycles (2months) was given to each patient. All of the women had previouslyreceived a minimum of three chemotherapy regimens includinganthracyclines (26/27) and taxanes (24/27). The patients were monitoredfor response to treatment using physical exams, tumor markers, andPET/CT fusion scanning. All patients were consistently monitored for anyclinical signs of toxicity or adverse effects.

Three patients had complete responses (11%), 13 patients had partialresponses (48%), resulting in an overall response rate of 59%. Sevenpatents had stable disease and four patients progressed. Overalltoxicity was acceptable, however 6 patients had side effects with onepatient being withdrawn from the treatment.

The combination of Abraxane® and Avastin® was very active in apopulation of heavily pre-treated women with metastatic breast cancer.The results demonstrated an objective clinical response rate of 59% (3complete response and 13 partial response).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is apparent to those skilled in the art that certainminor changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

What is claimed is:
 1. A method for treating breast cancer in anindividual, the method comprising: (a) determining hormone receptorstatus of estrogen receptor and progesterone receptor; (b) administeringto the individual an effective amount of a composition comprisingnanoparticles comprising paclitaxel and an albumin, and (c)administering to the individual an effective amount of epirubicin andcyclophosphamide, wherein the hormone receptor status of the individualis negative for both estrogen receptor and progesterone receptor.
 2. Themethod of claim 1, wherein the average diameter of the nanoparticles inthe composition is no greater than about 200 nm.
 3. The method of claim1, wherein the albumin is human serum albumin.
 4. The method of claim 1,wherein the weight ratio of the albumin and the paclitaxel in thenanoparticle composition is about 9:1 or less.
 5. The method of claim 1,wherein the individual is human.
 6. The method of claim 1, wherein thehormone receptor status of estrogen receptor and progesterone receptoris determined by immunohistochemistry.
 7. The method of claim 1, whereinthe breast cancer expresses HER2 (HER2+).
 8. The method of claim 1,wherein the dosage of paclitaxel in the nanoparticle composition is50-300 mg/m².
 9. The method of claim 1, wherein the method is fortreatment of breast cancer in a neoadjuvant setting.
 10. The method ofclaim 1, wherein the dosage of paclitaxel in the nanoparticlecomposition is 125 mg/m².
 11. The method of claim 1, wherein the averagediameter of the nanoparticles in the composition is no greater thanabout 150 nm.
 12. The method of claim 1, wherein the average diameter ofthe nanoparticles in the composition is about 130 nm.
 13. The method ofclaim 1, wherein the paclitaxel is coated with albumin.
 14. The methodof claim 2, wherein the weight ratio of the albumin and the paclitaxelin the nanoparticle composition is about 9:1 or less, wherein thepaclitaxel is coated with albumin.
 15. The method of claim 2, whereinthe method is for treatment of breast cancer in a neoadjuvant setting.16. The method of claim 2, wherein the dosage of paclitaxel in thenanoparticle composition is 50-300 mg/m².
 17. The method of claim 14,wherein the dosage of paclitaxel in the nanoparticle composition is50-300 mg/m².
 18. The method of claim 11, wherein the weight ratio ofthe albumin and the paclitaxel in the nanoparticle composition is about9:1 or less, wherein the paclitaxel is coated with albumin.
 19. Themethod of claim 12, wherein the weight ratio of the albumin and thepaclitaxel in the nanoparticle composition is about 9:1 or less, whereinthe paclitaxel is coated with albumin.